database connection failed! database connection failed! database connection failed! database connection failed! database connection failed!  126 indonesian journal of cancer chemoprevention, october 2018 issn: 2088–0197 e-issn: 2355-8989 curcumin analog pentagamaboronon-0-sorbitol inhibits cell migration activity of triple negative breast cancer cell line ratna dwi ramadani1, rohmad yudi utomo2, adam hermawan2,3, edy meiyanto2,3,* 1magister program of biotechnology, faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia 2cancer chemoprevention research center, faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia 3departement of pharmaceutical chemistry, faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia abstract mortality in cancer is primarily due to failure of metastasis prevention. one strategy to target the cancerous cell is boron neutron captured therapy which showed high affinity toward cancer cells and reported to have anti-proliferative as well as antimetastatic activities. cancer chemoprevention research center faculty of pharmacy universitas gadjah mada, has developed boron-containing substance namely pentagamaboronon-0 (pgb-0) which is known to exhibit anticancer activity towards breast cancer cell. the purposes of this research are focused to explore the anti-migratory activities of pgb-0-so against triple negative breast cancer cell. the mtt cytotoxicity assay of pgb-0-so against 4t1 breast cancer cell line were found to exert potential effect in dose-dependent manner with ic50 values of 39 µm. the study of cell migration inhibition using in vitro wound healing assays and gelatin zymography on highly metastasis breast cancer cell line 4t1, following the treatment of sub ic50 doses of pgb-0-so complex slightly inhibited cell migration through the inhibition of matrix metalloproteinase-9 expression. these findings suggest that pgb-0-so is potential as an anticancer agent. keywords : curcumin analogue, pgb-0-so, 4t1 cells, migration, mmp-9 submitted: july 27, 2018 revised: september 4, 2018 accepted: september 7, 2018 *corresponding author: edy_meiyanto@ugm.ac.id introduction breast cancer is the most common type of cancer causing mortality for women. in 2012, there were registered 1.67 million new cases of breast cancer mortality in women by 198,000 (ferlay, et al., 2012). it was estimated that approximately 10-15% of breast cancer was known to be triple negative breast cancer (tnbc) (dawood, 2010). this breast cancer subtype is positive-metastatic breast cancer (mbc) which have worse prognosis leading to aggressive disease. moreover, failure of metastasis prevention primarily caused mortality in breast cancer. the treatment of metastasis in breast cancer was conducted by chemotherapy, such as doxorubicin which performed strong cytotoxicity against cancer cells. despite its potent anticancer activity, doxorubicin had several limitations for long-term use including cardiotoxicity and chemoresistance (carvalho, et al., 2009; thorn, et al., 2011). in addition, low dose of doxorubicin induces 127 ramadani, et al, 2018 indones. j. cancer chemoprevent., 9(3), 126-133 epithelial-mesenchymal transition (emt) leading to metastasis on breast cancer cells (mbc) (bandyopadhyay, et al., 2010). hence, several antimetastatic agents had been developed to treat mbc. development of anti-metastatic agents as potential candidate of chemotherapeutic agents has been established over the years. patients characterized as triple negative breast cancer are treated with taxanes or platinum compounds (gavilá, et al., 2015). similar to doxorubicin, taxanes in low dose induced peripheral neuropathy in breast cancer (bhatnagar, et al., 2014), while platinum compound (cisplatin) induced emt in ovarian cancer (baribeau, et al., 2014). thus, the effective anti-metastatic agents need to be developed further. curcumin analogues based on benzylidine cyclopentanone backbone such as pentagamavunon-0 (pgv-0) and pentagamavunon-1 (pgv-1) exert potent cytotoxic (meiyanto, et al. (2006); nurulita, et al. (2006); dai, et al. (2007); dai, et al. (2011); hermawan, et al. (2011); meiyanto, et al. (2014) and anti-metastatic activities toward several types of breast cancer cells (putri, et al., 2016). pentagamaboronon-0 (pgb-0) is a novel curcumin analogue based on benzylidine cyclopentanone developed by faculty of pharmacy universitas gadjah mada. cytotoxicity of pgb-0 toward her2 positive breast cancer had been determined and showed to decrease her2 expression (utomo, et al., 2017). pgb-0 also performed anti-metastatic activity toward triple negative breast cancer cells (unpublished data). however, similar to curcumin, pgb-0 is less soluble in water. to improve solubility of pgb-0, we synthesized the complex form of pgb-0 with polyol sugar, sorbitol, namely pgb-0so. this study aims to develop pgb-0-so as novel anti-cancer agent especially through the inhibition of cell migration. 4t1 cells were used as a model of highly metastatic breast cancer cell line. possible anti-metastasis activities of pgb-0-so were analyzed by using scratch wound healing assay and gelatin zymography. the result of this study will be used for further experiment in order to develop novel anti-migratory agents from pgb-0-so. materials and methods chemicals pgb-0-so was synthesized by cancer chemoprevention research center, faculty of pharmacy, universitas gadjah mada. doxorubicin was purchased from sigma. cell culture 4t1 breast cancer cells were obtained from prof. masashi kawaichi, m.d., ph.d (nara institute of science and technology, naist, japan). the cells were maintained in dulbecco’s modified eagles medium (dmem) high glucose (sigma, st. louis, ca, usa) with 10% fbs (sigma), hepes, sodium bicarbonate, 1000 u/ml of penicilin-1000 u/ml of streptomycin and 0.5 µg/ml fungizone (gibco, new york, usa). scratch wound healing assay the 4t1 breast cancer cells were seeded 7.5x104 cells per well in 24 well-plate. cells were incubated for 24 hours until 80% confluent. media was removed and well was washed with 100 µl pbs (sigma). then cells were added with media contained 0.5% fbs for starvation and incubated for 22 hours. each well was scratched vertically by using yellow tip and treated with doxorubicin 10 nm as positive control, pgb-0-f, and combination of both compounds. the closures of cell migration were observed in 0, 18, 24 and 42 hours under inverted microscope (olympus, tokyo, japan) and captured by handphone (samsung, seoul, south korea). gelatin-zymography methods a total of 2 x 105 4t1 cells were planted in a 6-well plate with 2 ml of culture medium and incubated for 24 hours. the samples solution was carried out using culture medium containing  128 indonesian journal of cancer chemoprevention, october 2018 issn: 2088–0197 e-issn: 2355-8989 0.5% fbs. after incubation, the media is removed and washed with pbs 1 ml 1 times. cell then treated with various concentration of pgb-0-so and doxorubicin. estradiol was used to induce the expression of mmp-9. the cells then were incubated again for 48 hours. culture medium then collected in 1.5 ml microtube and centrifuged at 4oc and then take the supernatant. the sample has been obtained by loading the buffer and running in appropriate concentration electrophoresis. gel that has gelatin mold. the checking process with electrophoresis (v = 120v, i = 60a) was carried out for 130 minutes. after that, the gel is done and renaturation using a renaturing solution containing triton-x 100 to remove sds for 30 minutes. continued with incubation for 20 h using an incubation solution at 37oc. then, it should be stained with coomasie brilliant blue for 30 minutes, and through destaining using a destaining solution until the transparent blue band appears. data analysis scratch wound healing analysis was figured out by measuring the distance between scratch edges using imagej software then comparing the untreated and treated cells. data from multiple scratch within the same test group were analyzed using analysis of variance test to analyze the difference between experimental group. data were presented as mean ± s.d and analyzed by oneway anova. p < 0.05 was considered statistically significant. results and discussion anti-migratory effect of pgb-0-so and its combination with doxorubicin against 4t1 cell the purpose of this study is to develop a novel anti-cancer agent which especially plays role in inhibiting cancer cell migration. in this study, we used highly metastatic breast cancer cell model, 4t1, to explore the potency of pgb-0-so in inhibiting cell migration. low concentration of doxorubicin (dox) in this study was used to induce migration (bandyopadhyay, et al., 2010). scratch wound healing assay was conducted to observe cells migration following the treatment of several concentrations under ic50 value of 1/8, 1/4, and 1/2 of ic50, which are nontoxic and appropriate concentration to observe cell migration activity. the ic50 value of pgb-0-so against 4t1 cell was 40 μm. after 42 hours observation, dox increased the % closure up to 100% indicating the cell migration activity. on the other hand, single treatment of 1/2 ic50 pgb-0-so (20 μm) showed significant inhibitory activity against 4t1 cell line whereas the concentrations of 1/4 and 1/8 ic50 value (10 and 5 μm) of pgb-0-so showed unsignificant inhibitory anti-migratory activity compared to the untreated group. interestingly, combination of dox with all concentrations of pgb-0-so showed significant anti-migratory activity against doxorubicin-induced 4t1 cell (figure 1). the result needs to be confirmed with the expression of certain protein played role on cells migration or invasion. effect of pgb-0-so and its combination with doxorubicin against mmp-9 expression secondary cancer cells migration and invasion was tightly regulated by certain proteins especially matrix metalloproteinase 9 (mmp-9). role of mmp-9 on cells migration and invasion was to degrade extracellular matrix (ecm) around cancer cells (yabluchanskiy, et al., 2013; reunanen, et al., 2013). to observe possible effect of pgb-0-so on the decreasing expression of mmp-9, gelatine zymography was performed. low concentration of dox (10 nm) showed decreased expression of mmp-9 expression indicating the different possible effector of anti-migratory activity might be induced by dox. amalina, et al. (2017) reported that low dose of dox induced emt through rac1 independent-lamellipodia formation by which the initial progression of cancer cell migration. in contrast, both single treatment of pgb-0-so and 129 ramadani, et al, 2018 indones. j. cancer chemoprevent., 9(3), 126-133 figure 1. anti-migratory effect of pgb-0-so against highly metastatic, 4t1, cells migration. 4t1 cells (7.5x104 cells/well) were treated with pgb-0-f in the concentration as indicated in the figure, then subjected for scratch wound healing assay. a: the morphology of the cells after scratch and treated with pgb-0-so. observations were made after 18, 24 and 42 hour of treatment under an inverted microscope with magnification of 100x. b: the percentage of 4t1 cells closure after treatment. the area of the scratch were analyzed using imagej software then % closure was calculated in accordance with the procedures of the analysis (p<0.05). untreated dox 10 nm pgb-0-so 20 μm pgb-0-so 10 μm pgb-0-so 5 μm pgb-0-so 20 μm + dox pgb-0-so 10 μm + dox pgb-0-so 5 μm + dox 0 18 24 42 d ur at io n (h ou rs ) % c lo su re 40 20 0 100 80 60 untreated pgb-0-so 20 μm pgb-0-so 10 μm pgb-0-so 5 μm pgb-0-so 20 μm + dox pgb-0-so 10 μm + dox pgb-0-so 5 μm + dox dox 10 nm a b  130 indonesian journal of cancer chemoprevention, october 2018 issn: 2088–0197 e-issn: 2355-8989 its combination with dox showed the decreasing expression of mmp-9 protein in dose dependent manner (figure 2). hence, anti-migratory effect of pgb-0-so against 4t1 cell might be through the suppression of mmp-9 expression. discussion inhibition of tumor cell migration is crucial in the therapy and inhibition of cancer spread, especially in metastasis. thus, it is necessary to develop anti-migratory agent to overcome this situation. previous research showed that pgb0 exhibited anti-migratory effect as well as inhibited mmp-9 expression (unpublished data). mmp-9 is a family member of zincand calciumdependent endopeptidases, 88 kda protein which has numerous cell activities, involving in various figure 2. result of mmp-9 expression following the treatment of pgb-0-so, doxorubicin, and pgb-0-so combination with doxorubicin (48 hours). a: clear protein bands formed indicating the presence of mmp-9 protein. b: intensity quantification of mmp-9 band compared to the untreated groups. fo ld e xp re ss io n of m m p9 co m pa re d to th e un tr ea te d gr ou p 1.4 1.2 1.0 0.8 0.6 0.4 0.2 a b estradiol 40 nm doxorubicin 10nm pgb-0-so 5 μm pgb-0-so 10 μm pgb-0-so 20 μm + + + + + + + + + + + + + + + + + + mmp-9 physiological functions, such as cell-cell contact, tissue remodeling cell migration and cellular differentiation (yabluchanskiy, et al., 2013; vandooren, et al., 2013). other study using boron containing compound, phenylboronic acid showed that this compound has potency as selective inhibitor of cancer cell migration and viability without effecting non-tumorigenic cell lines (bradke, et al., 2008; mcauley, et al., 2011). the main purpose of this study is to explore the potential metastasis-inhibitory of pgb-0-so on triple negative breast cancer cells, 4t1. in this study, 4t1 cells were used as the model of human metastatic breast cancer cells because it is highly metastatic breast cancer cells. this cells can metastasize to liver, lung, bone and brain, making it a good model of human metastatic breast cancer (pulaski, et al., 2000; dupré, et al., 2007). 131 ramadani, et al, 2018 indones. j. cancer chemoprevent., 9(3), 126-133 doxorubicin is usually used as chemotherapeutic first-line treatment of several type of cancer especially triple negative breast cancer. prolonged use of doxorubicin showed toxicity effect such as cardiotoxicity and hepatotoxicity (pedrycz and kramkowska, 2016). on the other hand, another research reported that doxorubicin at low dose can enhanced cancer cell migration by inducing lamellipodia formation (amalina, et al., 2017). previous studies showed the migration inbitory activity of pgb-0-so (pentagamaboronon0-sorbitol) in 4t1 cells (unpublished data). pgb-0so showed cytotoxic effect on 4t1 cells with ic50 values of 39 µm (unpublished data). while pgb-0 has ic50 value 300 µm in 4t1 cells (unpublished data), and 270 µm in mcf-7/her2 cells (utomo, et al., 2017). in this present study, we also observed the inhibition of cancer cell migration as the one of parts of metastasis process by treatment of pgb0-so through scratch wound healing assay. cell migration is part of the metastasis process. based on the percent graph of 4t1 cell closure (figure 1a) the treatment of pgb-0-so with concentration 5 and 10 µm had demonstrated unsignificant migration inhibitory activity in all time course compared to the negative control group (untreated). on the other hand, 20 µm concentration of pgb-0-so began to affect significantly the inhibition of cell migration especially after 42nd hours. the treatment of doxorubicin 10 nm showed higher % closure than untreated cells, it means that doxorubicin induced cell migration. previous study reported that doxorubicin induced lamelipodia formation and cell migration in 4t1 and mcf-7/her2 cells (amalina, et al., 2017). furthermore, combination treatment of pgb-0so and doxorubicin showed inhibition of 4t1 cell migration 42 hours after incubation time. at this incubation time both single pgb-0-so 1/2 ic50 treatment and its combination with 10 nm doxorubicin showed significant differences with the difference in closing percentages compared to the control of cells without treatment. whereas when pgb-0-so treatment with doxorubicin 10 nm compared with a single treatment doxorubicin 10 nm resulted in lower closing percentage indicating the inhibitory activity of pgb-0-so. it has been reported in the breast cancer patients where there is a significant association between high mmp9 expression and poor survival (song, et al., 2013) so that mmp-9 expression would be potential therapeutic agents to inhibit development of cancer metastasis. curcumin analogue pgb-0-so showed tendency of inhibitory effect of mmp-9 activity in gelatin zymograph assay 48 hours after treatment. previous study also showed that pgb-0 inhibited mmp-9 expression (soon will be published). curcumin itself showed the inhibition of mmp-9 expression by inhibiting mitogenactivated protein kinase (mapk) phosphorylation (cao, et al., 2014). other research showed that simultaneous silencing of mmp-9 in breast cancer cells decreased the wound healing, migratory, invasive and adhesive capacity of the cells by increasing cell-cell adhesion and modulating emt genes (moirangthem, et al., 2016). conclusion pgb-0-so exhibits anti-migration effect against doxorubicin treatment cells. pgb-0-so also inhibits mmp-9 activity which has role in tumor invasion. references amalina, n.d., nurhayati, i.p., and meiyanto, e., 2017, doxorubicin induces lamellipodia formation and cell migration, indones. j. cancer chemoprevent., 8(2), 61–67. bandyopadhyay, a., wang, 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failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed! database connection failed!  7 indonesian journal of cancer chemoprevention, february 2019 issn: 2088–0197 e-issn: 2355-8989 expression of human erythropoietin containing 2 additional n-link in cho-k1 cells under different culture conditions adi santoso*, larasati, arizah kusumawati, popi hadi wisnuwardhani, ratih asmana ningrum, endah puji septisetyani research center for biotechnology, indonesian institute of sciences, cibinong, bogor 16911, indonesia abstract human erythropoietin (hepo) is a glycoprotein that regulates the formation of erythrocytes and mainly used in anemia patients. previously, we have reported the expression of modified human epo with 2 additional n-linked in mammalian cell cho-k1. the aim of this current research was to study the optimum condition for modified recombinant hepo (rhepo) production in cho-k1. to do this, several parameters of culture conditions were applied including antibiotic concentrations, seeding densities, time of incubations, fetal bovine serum (fbs) concentrations and cell culture media. the result showed that the presence of antibiotic g418 improved the expression level with the highest was at 1% of concentration. meanwhile, seeding density of 2–3x105 cells/6 cm dish and seven day of incubation time were the best condition for rhepo protein expression. from five different combination media used, f12 medium with 10% fbs gave the highest expression of rhepo protein. from this study was also found that at passage 16 the expression level was still increasing proving that the clone expressing the protein of our interest is promisingly stable. keywords : epo, erythropoietin, protein expression, cho-k1, optimation submitted: november 2, 2018 revised: november 26, 2018 accepted: november 28, 2018 *corresponding author: adi.santoso1960@gmail.com introduction as the main protein involved in the maintenance of red blood cell level in the body, the function of erythropoietin (epo) is highly regulated by the presence of oxygen in the body (krantz, 1991; lacombe, et al., 1998). the decrease of oxygen in tissues will enhance the production of epo in kidney (d’andrea, et al., 1989). thus, the production of red blood cell is influenced by the level of oxygen in the body. erythropoietin (epo) is a complex and intensely glycosylayted particle comprising of 165 amino acids. its molecular mass is 30.4 kda, but it relocates with an apparent size of 34-40 kda on sds-polyacrylamide gels. however, the correct molecular weight profoundly relies upon the level of glycosylation. this molecule contains three n-linked and one o-linked carbohydrate side chains with maximum may contains 14 residues of sialic acid. approximately, 40% of its molecular weight is contributed by its sugar partition (egrie, et al., 1986). the n-linked sugar side chains give off an impression of being fundamental for the formation of epo, limit clearance, increase stability, thus can 8 santoso, et al, 2019 indones. j. cancer chemoprevent., 10(1), 7-15 perform erythropoiesis normally in bone marrow (fried, et al., 1972; zanjani, et al., 1981; yin, et al., 2000). precedent study egrie, et al. (2001) had demonstrated that the expansion of sialic acid content of epo with the integration of 2-n linked can increase serum half-life and biological activity. insufficiency of epo becomes one of the main causes of cancer anemia, this makes the use of recombinant therapeutic protein epo becomes one of the solutions (kasper, et al., 1997). present study shows that epo is very beneficial to alleviate cancer-associated malignant anemia and can improve survival outcomes for patients with cancer (zhao, et al., 2017). mortality among patients with anemia is twice as high as that among those without anemia at three year post-cancer diagnosis (rice, et al., 1999; leng, et al., 1999., chung, et al., 2003). this indicates that the use of epo in treating cancer anemia can be very helpful. with numerous licenses for the principal biologicals derived from recombinant innovation are terminating. normally, biosimilars are becoming an increasingly consequential area of interest for the pharmaceutical industry globally and this likewise opens up open doors for creating nations to deliver their own particular biologic items (katherine, et al., 2014; blakstone, et al., 2013; declerck, et al., 2017). with the long haul objective of creating biosimilar epo, investigation of human epo molecule containing 2 extra n-linked in mammalian cell cho-k1 is in progress. we already reported the expression of this modified recombinant human epo (rhepo) in cho-k1 cells and its in vitro proliferative activity in tf-1 cells (santoso, et al., 2014). the objective of this current study is to obtain the optimum conditions of expression of hepo protein in mammalian cells cho-k1 using general commercial media. the optimation of culture conditions under study includes antibiotic concentrations, seeding densities, time of incubations, fetal bovine serum (fbs) concentrations and cell culture media. materials and methods cell culture and reagents the cho-k1 cells were obtained from prof. masashi kawaichi, nara institute of science and technology (naist), japan. the cells were cultured in nutrient mixture f-12 ham (f12) media (sigma n6658) in the presence of 10% of fetal bovine serum (fbs, sigma), 100u of benzylpenicillin and 100 μg of streptomycin (gibco, invitrogen). cho-k1 cells were transfected with plasmid j-epo, (pj-epo) containing 2 additional n links and the stable cells expressing rhepo were used for this study (santoso, et al., 2014). as much as 1% of antibiotic g418 (sigma, a1720) was added to the media just before use to maintain protein expression. the cell was cultured in an incubator with the condition of 5% co2 and 37oc temperature. unless otherwise stated, the number of cell seeded was 1.5x105 cells/6 cm dish. the cells were seeded in 5 ml media in 6 cm dish. optimation study for the optimation study, the procedure is described as follow: 1) antibiotic g418 concentrations: the cells were cultured with the addition of 0, 0.5, 1, 1.5 and 2% of antibiotic g418 in f12 medium plus 10% fbs, 2) seeding density: the cells were cultured for four days in f12 medium in the presence of 10% fbs, 1% antibiotic g418, and seeded as many as 2x105, 3x105, 4x105 and 5x105 cells/6 cm dish; 3) incubation time: the cells were cultured in f12 medium in the presence of 10% fbs with seeded cells as many as 2x105 cells/6 cm dish and incubated for 2, 3, 4, 5, 6 or 7 days in the presence of 1% antibiotic g418, respectively; and 4) media and its concentrations: the cells were cultured in cd-cho (100%), cd-cho/f12 (3:1)/2.5% fbs, sfm (100%), sfm/f12 (3:1)/2.5% fbs and f12/10% fbs media in the presence of 1% antibiotic g418.  9 indonesian journal of cancer chemoprevention, february 2019 issn: 2088–0197 e-issn: 2355-8989 sds-page and western blotting. the purity of the protein analyzed by sds/ page performed as previously described in a 12% separating gel with a 5% stacking gel using the mini-protean-3 apparatus (biorad, hercules, ca, usa). following electroporation, proteins were transferred to amersham hybond ecl (ge healthcare) by electroblotting. western blots were performed using polyclonal anti human epo antibody (sigma, st. louis, mo, usa) as the primary antibody and anti-rabbit igg alkaline phosphatase linked whole antibody (promega, madison, wi, usa) as the secondary antibody. the bands were detected by bcip/nbt color development substrate (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) (promega, madison wi, usa). results several techniques of transfection strategies have been established to steadily integrate vector dna into mammalian cells. previously, we also reported the optimization cationic lipid mediated transfection of pj-epo in cho-k1 cells (septisetyani, et al., 2012). the transfected cells were selected in view of aminoglycoside phosphotransferase (antibiotic g418) selectable. to evaluate the antibiotic g418 concentration in relation to rhepo expression and cell growth, several concentrations of antibiotic g418 were added to the f12 growth media in the presence of 10% fbs. data shows (figure 1a) that the rhepo protein bands present around 34-45 kda corresponded to the theoretical molecular mass for the epo that the exact molecular weight highly depends on the degree of glycosylation. as shown in figure 1a the rhepo protein expression was approximately the same at 0, 0.5 and 1.5% of g418 antibiotic concentration; and the highest is at 1%. however, at highest g418 antibiotic concentration (2%), the expression dropped. to examine the presence of antibiotic g418 on the growth of the cells, cell densities were observed after 4 days of incubation. the data reveals that at 0-0.5% of antibiotic g418, the cell densities are, approximately, the same (7x105 cells/6 cm dish). however, at higher concentration (1-2% of antibiotics), the cell density dropped, approximately, to 5 to 6x105 cells/6 cm dish, respectively. a b rhepo 0 0.2 0.4 0.6 0.8 c el l n um be r ( 10 )6 0 0.5 1 1.5 2 g418 concentration (%) figure 1. expression of rhepo protein in cho-k1 cell in several antibiotic g418 concentrations. the cells (200,000 cells/6 cm dish) were cultured for 4 days. a: western blot analysis of cho-k1 cells expressing rhepo. lane 1-5 correspond to 2, 1.5, 1, 0.5 and 0% of antibiotic concentrations, respectively. b: cell densities after four days of incubation. to analyze the stability of the cells to express rhepo protein, the cells were passaged for 5 times in the presence (1%) or absence of antibiotic g418. the results show that both cells were able to express rhepo; and the expression level increased with more passage numbers (figure 2a and b). the data showed that at passage 16, the expression level (in the presence of 1% antibiotic g418) was still increasing (figure 2c). 10 santoso, et al, 2019 indones. j. cancer chemoprevent., 10(1), 7-15 a b c rhepo rhepo rhepo figure 2. western blot analysis of cho-k1 cells expressing rhepo protein with or without antibiotic g418 in f12 medium in the presence of 10% fbs. a: the cells were cultured without antibiotic g418. lane 1-5 corresponds to 1-5 passage numbers, respectively. b: the cells were cultured in the presence of 1% antibiotic g418. lane 1-5 corresponds to 1-5 passage numbers, respectively. c: the cells were cultured in the presence of 1% antibiotic g418. lane 1-4 corresponds to cells with passage numbers 13-16, respectively. to further optimize the best condition for the cell to grow, next step for this optimation study is to evaluate the number of cell seeded. to do this, several numbers of cells were seeded in f12 + fbs 10% medium. following four days of incubation, the numbers of cells were counted and the protein hepo expression was analyzed using western blot. the data in figure 3a reveals that higher expressions were observed at seeding density of 2x105 and 3x105 cells/6 cm dish. while lower expressions were observed at higher seeding density (4x105 and 5x105 cells/6 cm dish). after 4 days of incubation, the cell densities linearly correlated with the number of cell seeded with the lowest and highest were when the cells were seeded at 2x105 and 5x105 cells/6 cm dish, respectively (figure 3b). a b 0 1.0 0.8 0.6 0.4 0.2c el l n um be r ( 10 )6 rhepo 2 3 4 5 seeding density (x10 cells/6 cm dish)5 figure 3. optimation of cho-k1 cell seeding density. a: western blot analysis of cho-k1 cells expressing rhepo. lane 1-4 correspond to cell densities of 200,000; 300,000; 400,000 and 500,000 cells/6 cm dish. b: cell densities after four days of incubation. to further evaluate for how long the cell has to be cultured to obtain the highest titer, 200,000 cells/6 cm media was cultured in f12 + fbs 10% medium. the cells were cultured and harvested everyday from day two to seven. as revealed in figure 4a, optimation of incubation time indicates a linear relationship between time of incubation and level of expression. the data shows that seven days of incubation time gave the highest level of expression. while, the lowest level of expression occurred at two days of incubation. the cell density data showed that cell number increases with the increase of incubation time; and the peak occurred at day four with the total number of cell was, approximately, 6x105 cells/6 cm dish. after four days of incubation, the cell density decreases sharply until at seven days  11 indonesian journal of cancer chemoprevention, february 2019 issn: 2088–0197 e-issn: 2355-8989 a b rhepo 0 0.8 0.6 0.4 0.2 c el l n um be r ( 10 )6 2 3 4 5 6 7 day figure 4. optimation of incubation time. cells were seeded at density 200,000 cells/6 cm dish and incubated for 2, 3, 4, 5, 6 or 7 days (1-6 respectively). a: hepo protein expression. b: cell density at harvesting day. of incubation. this result indicated that higher protein expression was obtained at longer incubation time without showing correlation with the cell density. with the advance of biologic medicines, manufacturing of protein therapeutic products with the use of cho cells has become the preferred choice. however, one of the main problems in this system is that most of mammalian cells have to grow in the presence of fbs. the presence of fbs may complicate the downstream process of the biologics of interest (reinhart, et al., 2013). thus, one of the goals of this study is to evaluate how the cells grow in several media including serum free media. to do this, the cells were grown in several media and followed by observation of its expression and cell density (figure 5). from the five different media used, sfm/f-12(3:1)/2.5% fbs and f12/10% fbs media gave, approximately, the same level of rhepo protein expressions (figure 5a), followed by cd-cho/f12 (3:1)/2.5% fbs and sfm 100%. to have a better understanding of the protein expression in relation to the cell growth, the cells grown in several media were counted. the data shows that the highest cell density was found when the cells grown in sfm/ f12 (3:1)/fbs 2.5% medium followed by cd-cho/f12 (3:1)/fbs 2.5%, f12/10% fbs, cd-cho (100%), and sfm (100%), respectively. a rhepo 0 1.0 0.8 0.6 0.4 0.2 1.2 1.4 c el l n um be r ( 10 )6 b c d -c h o 10 0% c d -c h o /f 12 (3 :1 )/ 2. 5% f b s sf m 1 00 % sf m /f 12 (3 :1 )/ 2. 5% f b s f1 2. 10 % f b s figure 5. expression of rhepo protein in cho-k1 cell line in several media. a: western blot analysis of cho-k1 cell conditioned media. lane 1-5 correspond to cd-cho 100%, cd-cho:f12 (3:1)/fbs 2.5%, sfm 100%, sfm:f12/fbs (3:1)/fbs 2.5% and f12/10%fbs in the presence of 1% antibiotic g418, respectively. b: cell density after four day incubation. discussion these days, cell culture has transformed into one of chief fields in present day biotechnology, especially in the field of human wellbeing. advancement of development in interdisiplinary fields, for example, human medication, cell science 12 santoso, et al, 2019 indones. j. cancer chemoprevent., 10(1), 7-15 and biotechnology has provoked the generation of biologics including vaccines, hormones and monoclonal antibodies (feng, et al., 2010; kunert, et al., 2016). nonetheless, since a large portion of these particles are glycoproteins, post-translational adjustment stages are exceptionally indispensable (matasci, et al., 2008). to oblige this condition the utilization of mammalian cell culture innovation is vital. with the use of limiting dilution technique, our previous work (septisetyani, et al., 2012) was able to clone cho-k1 cell transformant harboring codon optimized human epo gene containing five n-linked oligosaccharide chains. to optimize the growth and titer of the obtained clone, the cloned cell was observed in several conditions including antibiotic concentrations, type of media, times of incubation and numbers of cell seeded. one of antibiotic frequently used in mammalian cell line work is antibiotic g418 (feng, et al., 2010). to observe how the titer and growth of our cell transformant performed in the presence of antibiotic g418 concentration, several concentrations of this antibiotic were applied in cho-k1 cell transformant harboring codon optimized human epo gene. the result showed that the highest expression of rhepo protein occurred at 1% antibiotic concentration. interesting to note that at 0% of antibiotic concentration, as seen in figure 1a, the concentration of rhepo was relatively high. it is known that when transfected cells were kept under selective pressure (for example antibiotic g418 or methotrexate), the expression level of the gene of interest stably maintained at high level and the reverse occurred in the absence of selective pressure. thus, the higher protein expression at zero concentration of antibiotic g418 than that of 2% has to be taken caustiously at higher passage numbers. it is possible that with the increasing number of passage, in the absence of selective pressure, the expression of the protein will decline. in another word, the presence of selective pressure may be needed to keep the expression of protein of interest to remain high. in case of cell density, the data shows that the cell number drops at 1–2% of antibiotic g418 concentration. the decrease of the cell density, especially at the highest concentration (2%), was followed by the decrease of the protein expression (figure 1). consistent gene expression for specific period of time is the main indication of stable cell lines. unfortunately, obtaining stable cell lines can be exceptionally costly and tedious (feng, et al., 2010; bussow, et al., 2015). to analyze the stability of our cloned cells, the cells were cultured in the presence and absence of 1% of antibiotic g418 for 16 passages. the number of passage starts when the cells were treated with experimental treatment. as seen in figure 2, the cells were still strongly expressing the protein of our interest meaning that the cells were stable. interesting to note that at 16 passages, the trend of the expression was still increasing indicating that there is a high possibility that our gene of interest was inserted stably into the genome of the cell. seeding density in cell culture may play crucial role in subsequent cell division, particularly if that has to do with specific cell line (zhou, et al., 2011). following seeding, cell development in culture begins from the slack stage to the log stage where the development turns to grow exponentially (rolfe, et al., 2012). in adherent system, normally, cells quickly grow until no room left for expansion. at this point, proliferation greatly reduced and finally, ceases completely. to ensure the cell culture keeps on developing, the cell culture has to be passaged at the right time. considering the dynamic growth of cell explained above, optimum seeding density become important. to observe the optimum number of cell seeded, several cell densities (2, 3, 4 and 5x105 cells/6 cm dish) were seeded. the result showed that the highest expression was obtained when the cell was seeded at 2–3x105 cells/6 cm dish (figure 3). it is natural to expect that the highest seeding density would give the highest expression level. however, the data obtained showed that the highest seeding density (4x105 cells/6 cm dish) did not give the highest expression level. the likely  13 indonesian journal of cancer chemoprevention, february 2019 issn: 2088–0197 e-issn: 2355-8989 explanation of this data was that lower seeding density (2–3x105 cells/6 cm dish) gave the optimum condition for the cells to synthesize the rhepo protein. the inverse relationship between the protein expression and cell growth may reflect the balance nutrients requirement of cells at particular time. to some extent time of incubation of cell culture is related to the amount of protein being expressed. to observe for how long the cell has to be cultured in order to obtain the highest expression level, the cells were cultured for seven days. a linear relationship pattern was obtained between degree of expression level and time of incubation with the highest at day seven. having known the relation between expression and time of incubation, the relationship between time of incubation and cell density was then observed. the data showed that cell density increases with the increase of incubation time. after reaching the peak at day four the cell density decreases sharply with the lowest at seven days of incubation (figure 4). thus, the peak of cell density was not followed by the peak of protein expression. this data might show that it takes time for the cell to synthesize the protein of interest (rhepo) that finally peaked at day seven. in term of scalability, mammalian cells have historically been considered to be difficult to work due to factors such as low yield, serum requirement and medium complexity (reinhart, et al., 2013; carrillo, et al., 2015). the data (figure 5) shows that the presence of fbs is significant for the growth and expression of the protein of interest with the highest was at 10% of fbs. interesting to note that the expression level at fbs 10% (in f12) and 2.5% [in sfm/f12 (3:1)] are, aproximately, the same. however, the protein expression in 2.5% fbs [in cd-cho/f12 (3:1)] was little lower. the low protein expression in sfm (100%) and almost no expression at all in cd-cho (100%) were understandable since the attachment of cho-k1 cell required fbs (figure 5). while fbs can bolster the development of numerous types of cells, however, the type of its particular components that are critical for every individual cell have not yet been recognized thoroughly (shahdadfar, et al., 2005; valk, et al., 2017). mostly in the form of fbs, serum has been utilized in cell culture for a considerable length of time, and its utilization is really expanding. serum may not be the suitable enhancement to use in cell culture, particularly if the protein was planned for biopharmaceutical items for use in human (schroder, et al., 2004; zhang, et al., 2013). the use of serum free media has picked up acknowledgment and significance, nonetheless, building up a powerful serum free media has been troublesome (freshney, 2010; valk, et al., 2017), as demonstrated in this work that the utilization of 10% fbs gave the best results. expansion and competition of costly biologics have fuelled endeavors to enhance and upgrade cell culture media with the aim of achieving greatest outcomes and bringing down the expense of production (valk, et al., 2017; tatsuma and asayama, 2017). commonly now, due to the need to support high cell densities and efficiency, the media used for biologics production are without serum and have substantially higher supplement fixations than traditional media (carrillo, et al., 2015). cell growth and viability are paramount for biologics production. however, when serum is basically needed for some cells to grow, the growth and achieving the cells to express the protein of interest becomes very challenging (fontes, et al., 2014). as productivity and growth can have inverse relationship, and the cell limited resources are divided between those two functions, nutrient essential for growth may compete with those for protein production. to face this problem, initial and thorough optimation is extremely important for having the highest protein production possible in mammalian cell culture. conclusion this research has shown that to certain extent antibiotic g418 improves the hepo protein expression level with the highest was at 14 santoso, et al, 2019 indones. j. cancer chemoprevent., 10(1), 7-15 1% of concentration. seeding density and time of incubation affect the expression level with the best conditions are at 2–3x105 cells/6 cm dish and seven day of incubation time, respectively. medium f12 with 10% fbs proves to be the best medium for rhepo expression. however, the use of sfm/f12 (3:1) medium with lower concentration of fbs (2.5%) also gave promising result. observed for 16 passages, the expression level was still increasing indicating 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tissue eng., 17(21-22), 2603-2613. indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 182 cytotoxicity assay from fractions of hedyotis corymbosa extract against breast cancer cell line t47d rina andriyani 1* , chandra risdian 1 , zalinar udin 1 1 research center for chemistry, indonesian institute of sciences, jl. cisitu-sangkuriang, gedung 50, bandung-indonesia 40135 abstract drug discovery for cancer medication is the most important effort that researcher do at this time. indonesia bio diversities have possibility as a cancer medicine sources. finding a herbal medicine for cancer treatment is a first step to find a right cancer medicine in the future. this research has already completed for the earlier another research. some fractions of hedyotis corymbosa extract has been analyzed using sulforhodamine b method with uv wavelength 515 nm against t47d cell line, a human breast cancer. there are hexane extract, methylene chloride extract and ethyl acetate extract, and give inhibitory concentration 50 (ic50) of 33.45 µg/ml, 54.59 µg/ml and 52.58 µg/ml, respectively. ethanolic extract, itself has ic50 of 61.57 µg/ml, whereas ic50 value of cisplatinum is 9.63 µg/ml. there is a difference between the ethanolic extracts with the other fraction. keywords: breast cancer, herbal medicine, t47d, hedyotis corymbosa introduction drug discovery for cancer medication is the most important effort that researcher do at this time. indonesian bio diversities have possibility as a cancer medicine sources. finding a herbal medicine for cancer treatment is a first step to find a right cancer medicine in the future. hedyotis corymbosa is one of species from hedyotis (genus), rubiaceae (family), rubiales (ordo), dicotyledoneae (class), angiospermae (sub-division), and spermatophyta (division). they grow well on dry and sandy soil, along rivers and coasts and in the forests (ahmad, 2004). beside they widely grow in indonesia, they will widely found also in malaysia and india. rohaya ahmad, 2004, also said that previous studies on some hedyotis species have yielded indole alkaloids, anthraquinones, lignans, triterpenes, flavonoids as well as iridoids. in addition, three new iridoid glycosides are hedycorysides a-c has already found too (wei jiang, 2007). investigation to methanol extract of hedyotis corymbosa resulted that extract have some bioactivities. antibacterial activities, antiinflammatory, their radical scavenging, cytotoxic and hepatoprotective had studied (ahmad et al., 2004; sadasivan et al., 2006). this investigation, we aim to investigate cytotoxity ethanol extract and its fractions of hedyotis corymbosa. we use sulforhodamine b method against t47d cell line to know their inhibition concentration 50 (ic50). methods plant materials and preparation of extract the whole plants of hedyotis corymbosa were collected from indonesian medicinal and aromatic crops research institute. it determined by research center for biology, indonesian institute of sciences. *corresponding author email: titiksunarni@yahoo.co.id andriyani, et al., 2011 indones. j. cancer chemoprevent., 2(1), 182-186 183 the whole plants were washed, dried and powdered. maceration is done during 72 hours to gain crude extract, using ethanol technical quality as a solvent. more over to gain some fractions, from crude extract simultaneously extraction liquid-to-liquid using hexane, methylene chloride, and ethyl acetate. culture cell t47d (breast cancer) cell line was obtained from pharmacy department, gadjah mada university. cells were cultivated in dmem (gibco) medium supplemented with 10% v/v fetal bovine serum (sigma), 1% antibioticantimycotic (gibco). cells maintained in 25cm 3 flasks with 5 ml of dmem at 37°c with 5% co2. assay for cytotoxic activity cytotoxic assay was determined using sulforhodamine b method with uv wavelength 515 nm for measured. it is adapted from national cancer institute of america. samples concentration ranged between 100–3.125 µg/ml. each well filled with cell suspension in dmem amount of 10 4 cells/ml. as a control positive is using cisplatinum. the assay for each concentration of extract performed in triplicates and the culture plates incubate at 37 °c with 5% co2 for 24 hours. after 24 hours, old medium removed, washed with pbs solution, and added dmem fresh medium. samples with slightly concentration, added to the flasks, and then keep at 37°c with 5% co2. after 24 hours later, plates added cold 50% trichloroacetic acid, incubated at 4°c for 30 minutes, washed with tap water 5 times, and air dry plates until no standing moisture is visible. next step, cell staining. stain 100 µl/well of 0.4% srb in 1% acetic acid for 30 minutes. rinse off unbound dye with 1% acetic acid five times, air-dried. solubilize bound dye with 200 µl/well 10 mm tris base (ph10) for 5 minutes on a gyratory shaker. the measurement for optical density (od) at 515 nm used elisa plate reader. results range concentration ethanol extract of hedyotis corymbosa and its fractions showing percentage proliferation to t47d, between 3.125 100 µg/ml. the result is showing on table 1 below. the result show that 12.5 µg/ml of cisplatinum concentration as positive control equal to 100 µg/ml each concentration extracts of samples. percent proliferation of ethyl acetate extract given the best value than the others on the same concentration, it is 28.79%. table 1. percent proliferation of extract and its fractions of hedyotis corymbosa samples % proliferation 100 µg/ml 50 µg/ml 25 µg/ml 12.5 µg/ml 6.25 µg/ml 3.125 µg/ml ethanolic extract 31.93 51.67 65.36 73.63 92.16 82.04 hexane extract 34.43 40.27 43.69 71.06 91.87 94.16 mtc extract 34.93 49.32 73.06 88.09 75.98 81.39 ethyl acetate extract 28.79 50.53 76.76 93.65 46.69 62.22 cisplatinum 42.68 35.63 34.69 32.10 74.63 82.93 indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 184 linear regression used to analyze about correlation between concentration samples and proliferation percentage t47d cell line. base on linear regression analyzing, we can know a value of inhibition concentration 50 (ic50) from each samples. some graphics are shown below, from figure 1 to figure 5. hexane extract of hedyotis corymbosa given the best ic50 value, it is 33.45 µg/ml. all ic50 data are able to see in table 2. table 2. inhibition concentration 50 (ic50) of extract and its fractions of hedyotis corymbosa to t47d cells discussion the present study reports the cytotoxicity of hedyotis corymbosa extract and its fractions against t47d breast cell line. bioassay for the crude extract of hedyotis corymbosa has been reported have antioxidant, radical-scavenging, anti-inflammatory, cytotoxic, antibacterial activities and hepatoprotective (ahmad et al., 2004 and sadasivan et al., 2006). they reported that methanol extract of hedyotis corymbosa has cytotoxicity against cems-ss with cd50 value 21 µg/ml. cems-ss is a human t-lymphoblastoid cell line. our investigation resulted that ethanol extract of hedyotis corymbosa has cytotoxicity against t47d cell line, a human breast cancer, with ic50 value 61 µg/ml. ic50 value for its fraction shown a different activity. the value is better than the crude extract. it could be compounds in the crude extract have antagonism and compounds are able to synergism in fractions. sample inhibition concentration 50 (ic50) µg/ml ethanol extract 61.57 hexan extract 33.45 mtc extract 54.59 ethylacetat extract 52.58 cisplatinum 9.63 andriyani, et al., 2011 indones. j. cancer chemoprevent., 2(1), 182-186 185 figure 1. linear regression of ethanol extract figure 2. linear regression of hexane extract figure 3. linear regression of mtc extract figure 4. linear regression of ethylacetate extract figure 5. linear regression of cisplatinum indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 186 the former studied by the other researcher, hedyotis corymbosa contains some compounds. there are oleanolic acid, ursolic acid, sitosterol, stigmasterol, asperglaucide, indole alkaloids, anthraquinones, lignans, triterpenes, iridoid glycosides are hedycorysides a-c (liao et al., 1979; tong-ing ho et al., 1986; wei jiang, 2007). there is possibility that activity, which is shown, affected from one of compounds. it should be study further to know specific active compound that causing active as anticancer for this herb. conclusion we reported our studied to the crude extract (ethanol extract) of hedyotis corymbosa and its fractions have inhibition concentration 50 (ic50), 61.57 µg/ml and 33.45 µg/ml for hexane fraction; 54.59 µg/ml for methylene chloride; 52.58 µg/ml for ethyl acetate. acknowledgment the authors would like to thank the director of research center of chemistry for the fund provided under dipa grant. appreciation goes to ms. nur hidayati for the great technical support at extraction process. references ahmad, r., ali, a.m., israf, d.a., ismail, n.h., saari, k. and lajis, n.h., 2004, antioxidant, radical scavenging, antiinflamatory, cytotoxic and antibacterial activities of methanolic extracts of some hedyotis species, life sciences, 76(2005) 1953-1964. liou, w-c., lin, y-c., y-m. and chen, f-c. 1979, chemistry (taipei) 72. sadasivan, s., latha, p.g., sasikumar, j.m., rajashekaran, s., shyamal, s. and shine, v.j., 2006, hepatoprotective studies on hedyotis corymbosa (l.) lam., j ethnopharmacol., 106(2), 245-249. tong-ing, ho., chen, g.p., lin, y.c., lin, y.m. and chen, f.c., an anthraquinone from hedyotis diffusa, phytochemistry, 25(8), 1988-1989. wei jiang, li-sha kuang, ai-jun hou, min qian, and ji-zong li, 2007, iridoid glycosides from hedyotis corymbosa, helvetica chimica acta, 90(7), 1296-1301. indonesian journal of cancer chemoprevention, february 2010 issn: 2088–0197 e-issn: 2355-8989 32 leunca (solanum nigrum l.) herbs ethanolic extract increase cytotoxic activity of cisplatin on hela cervical cancer cells raditya prima istiaji 1 , maya fitria, larasati, fortunella tjondro, astrid ayu maruti, erna prawita setyowati, edy meiyanto* 1 cancer chemoprevention research center, faculty of pharmacy, universitas gadjah mada, yogyakarta *corresponding author email: meiyan_e@ugm.ac.id, cancer chemoprevention research center, faculty of pharmacy ugm, yogyakarta, indonesia,http://ccrc.farmasi.ugm.ac.id abstract cervical cancer is one of leading causes of cancer death in women in the developing countries. the use of cisplatin as chemotherapy agent in cervical cancer is known to cause side effects and also resistance for long-term uses. one of the strategies to prevent cervical cancer based on combination agents is being developed. leunca (solanum nigrum l.) has been revealed to inhibit growth of human cancer cells. therefore, it can be used in combination with cisplatin to reduce those side effects and prevent the occurrence of cell resistance. ethanolic extract of leunca herb (elh) and cisplatin were tested their cytotoxic effect on hela cervical cancer cell by using mtt assay to determine ic50 value. the combinationss of cisplatin-elh were tested to determine the combination index (ci value). the ic50 of elh and cisplatin on hela cells were 227 µg/ml and 17 µm. rrespectively. tthe study of combination resulted that almost all the index combinations were <0,9 showed the effect of synergism combination. the ooptimum concentration of combination was 1/8 ic50 cisplatin–1/8 ic50 elh. the results indicated that elh had a potency to be combination agent to enhance the activity of cisplatin on hela cervical cancer cells. therefore, further study on its molecular mechanism needs to be explored. key words: leunca (solanum nigrum l.), cisplatin, cytotoxic, combination agent, hela cells. introduction cervical cancer remains to be the one of the greatest killers of women in the worldwide. cisplatin as the chemotherapy in cervical cancer gave side effects like nephrotoxicity, neurotoxicity, ototoxicity, nausea, vomiting and resistant (cepeda et al., 2007; fuertes et al., 2003). the use of herbal medicines in combination with chemotherapy is one of the better ways in the treatment of advanced cancer cases because it can improve efficacy and reduce toxicity (liu et al., 2006) therefore, the research about alternative cancer therapy using herbal extract as combination with chemotherapy is important to be developed. leunca (solanum nigrum l.) is the plant that has anticancer activity. the previous in vitro studies showed that β-2-solamargine on leunca had cytotoxic effect on tumor cell lines ht-29 (colon), hct-15 (colon), lncap (prostate), pc-3 (prostate), t47d (breast), and mda-mb-231 (breast) (hu et al., 1999). the other studies, proved that solamargine could modulate the expressions of tnfrs and bcl-2 on h441, h520, h661 and h69 human lung cancer cells (liu et al., 2004). then solanine, a steroid alkaloid isolated from leunca was known inducing apoptosis in hepg(2) cells seems to be mediated by the inhibition of bcl-2 expression (ji et al., 2008). the researches above revealed that leunca had a potency to be anti-cancer agent. this study aims to explore the cytotoxic effect of leunca and its influence in combination with cisplatin on hela cervical cancer cells. *corresponding author email : meiyan_e@ugm.ac.id, http://ccrc.farmasi.ugm.ac.id mailto:meiyan_e@ugm.ac.id mailto:e@ugm.ac.id http://ccrc.farmasi.ugm.ac.id/ istiaji, et al., 2010 indones. j. cancer chemoprevent., 1(1), 32-37 -4567 33 methods ethanolic extract of leunca herb (elh). leunca herb dried powder was harvested and determinated by balai besar penelitian dan pengembangan tanaman obat dan obat tradisional (b2p2toot), tawangmangu, central of java. then the herbs were extracted with maceration method by using ethanol 70% (1:10). cell culture. hela cell line was obtained from prof. tatsuo takeya (nara institute of science and technology, japan) and grown in dulbecco’s modified eagle’s medium (dmem; gibco) supplemented with 10% fetal bovine serum (fbs; gibco) and 1% v /v penisilin-streptomisin (gibco) at temperature 37 °c and with a flow of 5% co2. cytotoxic assay with mtt method. hela was planted in 96-well plates with 5x10 3 cells/well and divided into control and treatment group. final concentrations of elh were 1, 5, 10, 25, 50, 100 and 250 µg/ml. 10 mg of elh was dissolved in dimethyl sulfoxide (dmso) then diluted in culture medium until final concentrations. concentrations of cisplatin were 1, 2, 5, 10, 20, 30 and 50 µm. after 24 h incubation, culture medium was removed and cells were washed using pbs (sigma). 5 mg/ml of mtt on pbs (sigma) was diluted by dmem (1:10) and 100 µl of it was added into each well. after incubate for 3 hours the reaction was stopped by sodium dodecyl sulfate (sds) 10% in hcl 0,1 n. after that, the plate was incubated for one night in room condition at dark place. to make sure the formazan dissolve, the plate was shaked for 10 minutes and measured the absorbance using elisa reader at wave length of 595 nm. the concentration applied on combination chemotherapy of elh was referred to ic50 value of each compound. the final combination concentrations of elh were 30, 60, 90 and 120 µg/ml, and for cisplatin concentration were 2, 4, 6 and 8 µm. statatistical analysis and data interpretation. absorbances measured from cytotoxic assay were analyzed by excell ms office and semi-log analysis (spss 11.5) to obtain ic50 value (doyle and griffiths, 2000). the combination index/ci (table i) was calculated based on equation below : note: d1 : combination concentration of for elh d2 : combination concentration for of cisplatin dx1 : concentration of elh in single dose that could inhibit the hela cells growth at the same point with combination concentration dx2 : concentration of cisplatin in single dose that could inhibit the hela cells growth at the same point with combination concentration (reynolds and maurer, 2005). table 1. interpretation of ci values ci value interpretation < 0,1 very strong synergistic effect 0,1 0,3 strong synergistic effect 0,3 0,7 synergistic effect 0,7 0,9 moderate synergistic effect 0,9 1,1 nearly additive effect 1,1 1,45 moderate antagonist effect 1,45 – 3,3 antagonist effect >3,3 very strong antagoni effect st istiaji, et al., 2010 indones. j. cancer chemoprevent., 1(1), 32-37 -4567 34 results cytotoxic effect of leunca herb etanolic extract on hela cells. the result showed that elh has cytotoxic effect on hela cells with ic50 value ofwas 227 µg/ml (figure 1a) while for cisplatin has the ic50 value was of 17 µm (figure 1b).this ic50 value showed that elh has the potency to inhibit hela cells growth. therefore, leunca could be used as a combination agent for cervical cancer treatment. (a) (b) figure 1. the cytotoxic effect of hela cells. treatment of elh (a) and cisplatin (b) the data were obtained by mtt assay with the incubation of cells for 24 h at temperature of 37°c and flow of 5% co2. each dot represent the means + sd from 3 replication. the combination effect of leunca herb etanolic extract and cisplatin on hela cells. despite its success, cisplatin has several disadvantages, like side effects and also the occurrence of resistance. therefore, the combination of chemotherapeutic agent and herb extract expected to increase the chemotherapeutic agent activity and also inhibit the occurrence of resistance. the parameter of combination effect was ci value,which used to assess the degree of combination effect in each concentration of combination. the combination of elh–cisplatin gave synergistic effect because almost all the ci value were under 0,9 (fig 2&3, table 2). the combination of elh-cisplatin that gave the biggest synergistic effect consisted of 30 µg/ml elh 2 µm cisplatin and 90 µg/ml elh 2 µm cisplatin. this combination gave ci value of 0,58. the viability data showed that the combination of elh-cisplatin (120 μg/ml-8 µm) inhibited the growth of hela cells until 46 %. this was higher than result given by single dose of each agent on the same concentration. istiaji, et al., 2010 indones. j. cancer chemoprevent., 1(1), 32-37 -4567 35 figure 2. combination effect of elh-cisplatin on hela cells morphology. the picture was obtained by mtt assay with the incubation of cells for 24 h at temperature of 37°c and a flow of 5% co2. (a) cell control ; (b) elh 250 µg/ml; (c) ciplatin 8 µm; and (d) 120 µg/ml elh2 µm ciplatin. normal hela cells ( ) and morphological changes of cells ( ) figure 3. the ci values of elh-cisplatin combination on hela cells. the data were obtained by mtt assay with the incubation of cells for 24 h at temperature of 37°c and with a flow of 5% co2. istiaji, et al., 2010 indones. j. cancer chemoprevent., 1(1), 32-37 -4567 36 table ii. ci values of elh-cisplatin combination. the bolds indicate ci value < 0,9 that have synergistic effect. the data was obtained by mtt assay. cisplatin (µm) leunca herb extract (µg/ml) 30 60 90 120 2 0,58 0,64 0,58 0,68 4 0,86 0,67 0,70 0,74 6 0,90 0,84 0,78 0,82 8 0,91 0,92 0,86 0,89 discussion this study showed that elh has cytotoxic activity and a synergistic effect in low concentration combination with cisplatin against hela cervical cancer cells. the mechanism of cancer cell growth inhibition occurs through many pathways such as apoptosis. apoptosis is the process of programmed cell suicide in the normal condition, it’s done to repair the broken tissues (hanahan et al., 2000). apoptosis occurs by the down-regulation of anti-apoptotic proteins such as bcl-2. based on the previous studies, leunca contains solanine and β-solamargine that able to decrease the expression of protein bcl-2 (liu et al., 2004; ji et al., 2008). expression of bcl-2 protein as an antiapoptosis protein can prevent cell death via apoptosis. therefore its decreased expression could induce the cancer cells death (pandanilam, 2003). in addition, solamargine or solanine could influence the upstream of the bcl-2, then inhibits the transcription of bcl-2 protein. finally, apoptosis can occur. our study proved that elh increased the cytotoxic activity of cisplatin on hela cervical cancer cells. therefore, study about molecular mechanism of active compound found in leunca herb ethanolic extract that induce the cisplatin cytotoxic activity needs to be explored. conclusion the conclusion of this study showed that combination between elh and cisplatin gave synergistic effect on hela and induced the cytotoxic activity of cisplatin on hela cervical cancer cell. references cepeda, v., miguel, a.f., josefina, c., carlos alonso, celia, q. and jose, m.p., 2007, biochemical mechanisms of cisplatin cytotoxicity, anti-cancer agents in medicinal chemistry, 7(3), 3-18. doyle, a. and griffiths, j.b., 2000, cell and tissue culture for medical research, john willey and sons ltd., new york. fuertes, m.a., alonso, c. and perez j.m., 2003, biochemical modulation of cisplatin mechanisms of action: enhancement of antitumor activity and circumvention of drug resistance, chem rev., 103, 645-62. hanahan, d. and weinberg, r.a., 2000, the hallmarks of cancer, cell, 100, 57-70. hu, k., kobayashi, h., dong, a., jing, y., iwasaki, s. and yao, x., 1999, antineoplastic agents. iii: steroidal glycosides from solanum nigrum, planta med., 65(1), 35-8. ji, y.b., gao, s.y., ji, c.f. and zou, x., 2008, induction of apoptosis in hepg2 cells by solanine and bcl-2 protein, j ethnopharmacol, 115(2), 194-202. liu, l.f., liang, c.h., shiu, l.y., lin, w.l., lin, c.c. and kuo, k.w., 2004, action of solamargine on human lung cancer cells-enhancement of the susceptibility of cancer cells to tnfs, febs lett. 577(1-2), 67-74. liu yi, rui wang, gen-quan qiu, ke-jun nan, and xi-cai sun, 2006, inhibitory effect of fuzheng yiliuyin in combination with chemotherapeutics on human gastric carcinoma cell strain, world j gastroenterol, 12(25), 4071-4073. file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez javascript:al_get(this,%20'jour',%20'planta%20med.'); file:///c:/sites/entrez file:///c:/sites/entrez file:///c:/sites/entrez file:///c:/sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez file://sites/entrez javascript:al_get(this,%20'jour',%20'febs%20lett.'); istiaji, et al., 2010 indones. j. cancer chemoprevent., 1(1), 32-37 -4567 37 padanilam, b.j., 2003, cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis, am j physiol renal physiol., 284, f608– f627. reynolds and maurer, 2005, evaluating response to antineoplastic drug combination in tissue culture models in methods in molecular medicine, vol. 110, humana press inc., totowa, nj. indonesian journal of cancer chemoprevention, june 2015 issn: 2088–0197 e-issn: 2355-8989 58 brazilein increased cytotoxic activity of doxorubicin on mcf-7/dox cells ni putu linda laksmiani 1* , ratna asmah susidarti 2 , and edy meiyanto 2 1 department of pharmacy, faculty of mathematics and science, universitas udayana, bali, indonesia 2 faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia abstract brazilein is a compound obtained in a large amount from the dried heartwood of secang (caesalpinia sappan l.). brazilein has strong cytotoxic effect in several cancer cell lines. this research was designed to evaluate the cytotoxic effect of brazilein and its combination with a chemotherapy agent, doxorubicin on mcf-7/dox breast cancer cells. in the cytotoxicity assay, mcf-7/dox cells were cultured in the presence of brazilein solely and in combination with doxorubicin for 24 hours and cell viability was evaluated by using mtt assay. mtt assay showed a dose-dependent inhibition of cell proliferation with ic50 value of 37 µm. brazilein increased doxorubicin’s cytotoxic activity on mcf-7/dox cells. both of single treatment with different concentration of brazilein 12.5 and 25 m or doxorubicin 0.8 and 1 m gave cell viability percentage above 80%, but combination of them led to decrease the cell viability percentage significantly. based on this research, it can be concluded that brazilein is potential to be developed as a co-chemotherapy agent on breast cancer cell that have been resistant to doxorubicin. futher study must be held to evaluate its molecular mechanism. keywords : brazilein, doxorubicin, mcf-7/dox, cytotoxic. introduction breast cancer is the first leading cause of cancer-related-deaths among women worldwide (acs, 2012). the high mortality rate indicates that chemotherapy has not been able to overcome cancer disease. one of the chemotherapeutic agents that is common to be used in breast cancer therapy is doxorubicin. however, several problems come up after the use of doxorubicin as a chemotherapeutic agent, such as its toxicity to normal tissues, severe side effects, and developed resistance. the side effects that usually arise are cardiomyopathy, congestive heart failure, and immunosupression. hence, strategies and development of breast cancer treatment should be pursued. one strategy that will be evaluated in this research is the development of phytochemicals to inhibit cancer cells’ growth as a chemopreventive agent and to reduce the problem faced in cancer treatment with chemotherapy, especially in breast cancer. there are a lot of medicinal plants that are potent to be used as chemopreventive agent. one of them is secang (caesalpinia sappan l.). secang has been traditionally used as coloring agent in foodsand beverages. besides, secang has a lot of pharmacological effects, especially in cancer. several phenolic compounds are isolated from c. sappan, such as homoisoflavonoid protosappanin a, protosappanin b, 4-o-methylsappanol, caesalpin j, brazilin, and brazilein (lim, et al., 1997). brazilin and brazilein are the major compound of this plant proven to be responsible for its cytotoxic effect on cancer. brazilein and brazilin (fig. 1) have cytotoxic effect on lung cancer cells, nasopharyngeal cancer cells, and prostate cancer cells with ic50 value of 5-18 µm (yen, et al., 2010). *corresponding author e-mail: lindalaksmiani@gmail.com laksmiani, et al., 2015 indones. j. cancer chemoprevent., 6(2), 58-63 59 (a) (b) figure 1. brazilein (a) and brazilin (b) brazilein inhibits survivin protein and induces apoptosis in hepg2 hepatocellular carcinoma cells (zhong, et al., 2009). this study was conducted to evaluate the cytotoxic effect of brazilein, both alone and in combination with doxorubicin on mcf-7/dox cells by using mtt assay. materials and methods sample collection brazilein was isolated from secang (caesalpinia sappan l.). secang was obtained in the form of dried heartwood powder from balai besar penelitian dan pengembangan tanaman obat dan obat tradisional (bbpptoot) in tawangmangu, indonesia. cell culture the mcf-7/dox cells was obtained from cancer chemoprevention research center (ccrc). the cells were routinely cultured in dmem supplemented with 10% fetal bovine serum (fbs) (sigma-aldrich, usa) at 37°c in a 5% co2 atmosphere, 1% penicillinstreptomycin, and 0.5 % fungizone. subcultures were obtained after treatment with 0.05% trypsin (gibco, auckland) in phosphate buffer saline (pbs). cytotoxic assay exponentially growing cells were seeded on 96-well plates at 1×10 4 cells per well and incubated for 24 hours prior to addition of drugs. test compounds were initially dissolved in dmso or h2o to make stock solution and then diluted with medium. following a 24 hours incubation at 37°c, 5% co2, 100 μl of various concentrations of brazilein were added in each well in triplicates and cells were further incubated for 24 hours. after 24 hours of incubation at 37°c, the medium was removed, and 100 μl of mtt reagent (1 mg/ml) in medium was added to each well. the plates were incubated at 37°c for 4 hours. at the end of the incubation period, the supernatant was removed, 10% sds 0.01n hcl (100 μl) was added to each well, and plates were shaken gently for 15 minutes. after an overnight incubation at 37˚c, the metabolized mtt product dissolved was quantified by reading the absorbance at λ 595 nm using an elisa reader (bio-rad). absorbance was then calculated in order to get the number of viable cells. to determine cell viability, percentage of cell viability was calculated as: the ic50 value is defined as the drug concentration required to inhibit cells growth by 50% of the control value. result and discussion effect of brazilein on mcf-7/dox cells growth the cytotoxicity of brazilein on mcf7/dox cells was determined by using mtt laksmiani, et al., 2015 indones. j. cancer chemoprevent., 6(2), 58-63 60 assay. the ic50 value was 37 µm (fig. 2). the mtt assay result demonstrated that brazilein had cytototoxic effect in mcf-7 cells. this effect was supported by the morphological change such as shrunken cell nuclei and membrane blebbing in some cells. the cytotoxic effect of brazilein in combination with doxorubicin must been held to evaluate the potency of brazilein as co-chemoterapy agent. figure 2. the cytotoxic effect of brazilein on mcf-7/dox cells. mcf-7/dox cells (1×104 cells/well) were seeded on 96 wells plate. the cells were treated with brazilein for 24 h. after 24 hours, cells were added by mtt reagent to calculate the absorbance which represent viable cells. (a) diagram of mcf7/dox cells viability after 24 hours brazilein treatment. mcf-7/dox cells morphology of (b) cell control; (c) after 24 hours 25 µm brazilein treatment; (d) 50 µm brazilein treatment. observation was done by using converted microscope with 400 x magnification. cell viability profile was shown from average ± standard of error (se) of 3 experiment. the normal cell morphology showed by the bold arrow ( ), meanwhile the change in cell morphology showed by the thin arrow ( ). (b) (c) (d) (a) laksmiani, et al., 2015 indones. j. cancer chemoprevent., 6(2), 58-63 61 brazilein increased doxorubicin’s cytotoxicity on mcf-7/dox cells in order to assess the increasing of doxorubicin's citotoxicity by using brazilein as co-chemotherapy agent in mcf-7/dox cells, the combinational cytotoxicity assay was conducted by using mtt assay. anova test (p>0.05) showed that the cytotoxic effect between brazilein and doxorubicin alone and in combination had significant difference. mtt assay result showed that brazilein increased the cytotoxic activity of doxorubicin on mcf7/dox cells (fig. 3). brazilein combination with doxorubicin on mcf-7/dox cells treatment showed increased the sensitivity of the cells. strong efficacy of brazilein in enhancing doxorubicin’s cytotoxicity may occur through several molecular mechanisms, one of them is by the apoptosis induction mechanism. it has already been reported that the cytotoxic effect of brazilein on hep g2 cells occured via apoptosis induction and survivin protein expression suppression (zhong, et al., 2009). figure 3. the cytotoxic effect of combination between brazilein and doxorubicin on mcf-7/dox cells. mcf-7/dox cells (1×104 cells/well) were seeded on 96 wells plate. the cells were treated with brazilein for 24 h. after 24 hours, cells were added by mtt reagent to calculate the absorbance which represent viable cells. diagram of combination cytotoxic effect of brazilein and doxorubicin (a). mcf7/dox cells morphology after 24 hours brazilein treatment, control cell (b); brazilein 25 µm (c); doxo 1 µm (d); brazilein 25 µm + doxo 1 µm (e) ( ) normal cell morphology; ( ) morphology cell changing. (a) (b) (c) (d) (e) laksmiani, et al., 2015 indones. j. cancer chemoprevent., 6(2), 58-63 62 long term use of doxorubicin causes severe side effect such as toxicity in normal cells and cancer resistance. it is most desirable to have more effective treatment by finding cochemotherapeutic drugs. co-chemotherapy is a cancer therapy strategy by combining natural agent or synthetic product with chemotherapeutic drugs. this strategy may reduce the side effect and toxicity of drugs. brazilein is one of the natural agents that had been isolated from ethyl acetate fraction of secang (caesalpinia sappan l.) by vacuum colom chromatography, yielding reddish crystal. brazilein inhibits mcf-7/dox cells’ proliferation with ic50 value of 37µm. according to teng, et al. (2005), compounds with ic50 values below 50 µm had a potent cytotoxicity against cancer cells, meaning that brazilein performed as a potent chemopreventive agent. the cytotoxic effect of brazilein on mcf-7/dox cells might be caused by cell cycle arrest or apoptosis induction. therefore, futher study must be conducted to evaluate the molecular mechanism that contribute to brazilein’s potency as chemopreventive agent against mcf-7/dox cells by using in vitro assay. based on ccrc unpublished data (2012), on hela cervical cancer and widr colorectal cancer cells, brazilein’s ic50 value were 61 µm and 243 µm, respectively. the different potency of brazilein in different cells is related to the characterization of each cell. mcf-7 cells were characterized with overexpression of bcl-2 and mutated caspase 3. in hela cells, p53 was inactive because of degradation, while overexpression of cox-2 is the characterization of widr cells. futher study had been done to investigate the increasing cytotoxic activity of doxorubicin using brazilein as co-chemoterapeutic agent by mtt assay. brazilein demonstrated strong efficacy as co-chemotherapeutic agent when combined with doxorubicin. lower doses of doxorubicin being used in combination with brazilein gave cytotoxic activity as potent as the doses used in single cytotoxicity assay. different mechanism of doxorubicin and brazilein contributes to the combinational cytotoxic effect of both of them. doxorubicin interacts with dna by intercalation and inhibits dna topoisomerase ii. based on previous study, brazilein was cytotoxic on mcf-7 cells by inducing apoptosis through suppression of survivin protein expression (tao, et al., 2011). survivin is the smallest member of the mammalian iap (inhibition of apoptosis) family that regulates cell death and cell cycle arrest (fortugno, et al., 2002; altieri, 2006). these molecular mechanism might be occurred by suppression of survivin protein expression through inhibition of its upstream protein, her-2 and ikk. inhibition of her-2 and ikk could suppress the pgp expression that correlated to resistance breast cancer cell induced by chemotherapeutic agent, doxorubicin (siddiqa, et al., 2008; gilmore, 2006). the overall results showed the combination of doxorubicin and brazilein have a potency for breast cancer therapy especially in mcf-7 cells that have been resistant to doxorubicin. effectiveness of combination therapy between brazilein and doxorubicin increased sensitivity of doxorubicin and induction apoptosis mechanism. brazilein can be development as an co-chemotherapy agent with doxorubicin. futher study must be established to evaluate the molecular mechanism of brazilein. conclusion brazilein was potential as doxorubicin co-chemotherapeutic agent on mcf-7/dox breast cancer cells with the ic50 value of 37 µm. acknowledgement this research was supported by cancer chemoprevention research center (ccrc) faculty of pharmacy universitas gadjah mada, yogyakarta (indonesia); balai besar penelitian dan pengembangan obat dan obat tradisional laksmiani, et al., 2015 indones. j. cancer chemoprevent., 6(2), 58-63 63 (bbpptoot), tawangmangu (indonesia); lembaga ilmu pengetahuan indonesia (lipi), serpong-banten (indonesia). references altieri, d.c., 2003, validating survivin as a cancer therapeutic target, nat. rev. cancer, 3(1), 46-54. american cancer society (acs), 2011, breast cancer facts & figures 2011-2012, atlanta: american cancer society, inc. fortugno, p., wall, n.r., giodini, a., o'connor, d.s., plescia, j., padgett, k.m., et al., 2002, survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function, j. cell sci., 115(3), 575-585. gilmore, t. d., 2006, introduction to nfkb: players, pathways, perspectives, oncogene, 25(51), 6680-6684. lim, d.k., choi, u., and shin, d.h., 1997, antioxidative activity of some solvent extract from caesalpinia sappan linn, korean j. food sci. technol., 28(1), 77−82. siddiqa, a., long, l. m., li, l., marciniak, r.a. and kazhdan, i., 2008, expression of her-2 in mcf-7 breast cancer cells modulates anti-apoptotic proteins survivin and bcl-2 via the extracellular signal-related kinase (erk) and phosphoinositide-3 kinase (pi3k) signaling pathways, bmc cancer., 8, 129. tao, l.y., li, j.y. and zhang, j.y., 2011, brazilein induced cells apoptosis in human breast cancer mcf-7 and its mechanism, j. sun yat-sen univ., 32, 449-453. teng, w.y., huang, y.l., shen, c.c., huang, r.l., chung, r.s, and chen, c.c., 2005, cytotoxic acridone alkaloids from te stem bark of citrus maxima, j. chinese chem. soc., 52(6), 1253-1255. yen, c., goto, k.n., hwang, t.s., wu, p.c., natschke, s.l.m., lai, w.c., et al., 2010, total synthesis and evaluation of brazilein and analogs as antiinflammatory and cytotoxic agents, bioorg. med. chem. lett., 20(3), 1037– 1039. zhong, b., wu, y.j., pan, s. and zheng, 2009, brazilein inhibits survivin protein and mrna expression and induces apoptosis in hepatocellular carcinoma hepg2 cells, neoplasma, 56(5), 387-392. indonesian journal of cancer chemoprevention, october 2015 issn: 2088–0197 e-issn: 2355-8989 89 cytotoxic and apoptotic-inducing effect of fraction containing brazilein from caesalpinia sappan l. and cisplatin on t47d cell lines prisnu tirtanirmala, annisa novarina, rohmad yudi utomo, raisatun nisa sugiyanto, riris istighfari jenie, edy meiyanto* cancer chemoprevention research center (ccrc), faculty of pharmacy, universitas gadjah mada, indonesia abstract anticancer activity of secang’s heartwood (caesalpinia sappan l.) is based on its main compound: brazilin and brazilein. brazilin, brazilein, and other compounds such as caesalpiniaphenol can affect proteins that have a role in apoptosis. in this study, we observed cytotoxic activity of fraction containing brazilein (fcb) alone or in combination with chemotherapeutic agent, cisplatin and the ability of the combination to induce apoptosis in t47d breast cancer cell lines. cytotoxicity assay was determined using mtt assay, whereas the detection apoptosis induction was conducted using flow cytometry using annexin-v and propidium iodide. fcb and cisplatin showed cytotoxic effect on t47d cells with ic50 value of 68 µg/ml and 16 µm, respectively. combination of fcb and cisplatin result synergistic combination at the concentration ratio of 1/2 ic50 with ci value of 0.66. its combination also able to induce apoptosis on t47d cell population 13% larger than the single treatment. based on this study, we conclude that fcb is able to enhance the cytotoxic effects of cisplatin by inducing apoptosis. keywords: caesalpinia sappan l., cisplatin, apoptosis, breast cancer introduction among women, breast cancer has the highest cancer incidence in seven asian countries including indonesia. cisplatin is a chemotherapeutic agent that commonly used in the treatment of various types of cancer, including breast cancer (tsimberidou, et al., 2009; dhar, et al., 2011). however, many anticancer drugs have narrow therapeutic index, so they can lead to multidrug resistance (mdr) (ismael, et al., 2008). they also cause side effects such as nephrotoxicity, neurotoxicity (milosavlievic, et al., 2010), and others. the use of co-chemotherapeutic agent combined with chemotherapeutic agent can improve the effectiveness of cancer therapy as well as reduce side effects (sharma, et al., 2004). one of the plants that potentially developed as an co-chemotherapeutic agent is secang’s heartwood (caesalpinia sappan l.). previous research proved that the main component of phenolic caesalpinia sappan l., is homoisoflavonoid like brazilein, chalcone, protosappanin and brazilin (yan, et al., 2005; shimokawa, et al., 1985; washiyama, et al., 2009). brazilein has cytotoxic effects on skin cancer cells to induce apoptosis through caspase-3-dependent (liang, et al., 2013). this study observed the cytotoxic effect and apoptosis induction of fraction containing brazilein (fcb) and its combination with cisplatin against t47d breast cancer cells. by doing this research, the results is expected to be a reference for further research in order to explore compound/active fraction of secang’s heartwood as cisplatin co-chemotherapeutic agent in the breast cancer treatment. materials and methods sample preparation fcb was obtained from the collection of cancer chemoprevention research center (ccrc), universitas gadjah mada. cisplatin (wako) was used as chemotherapeutic agent with concentration 1 mg/ml. *corresponding author email: meiyan_e@yahoo.com tirtanirmala, et al., 2015 indones. j. cancer chemoprevent., 6(3), 89-96 90 both fcb and cisplatin were diluted in various concentration using dulbecco's modified eagle medium (dmem) high glucose culture medium before treated to the cell. fcb was dissolved first in dimethyl sulfoxide (dmso) as co-solvent with final concentration 5 mg/ml. cells culture human breast cancer t47d culture cells were collection of cancer chemoprevention research center (ccrc), universitas gadjah mada. the cell line was kindly given by prof. kawaichi, nara institute of science and technology (naist), japan. chemicals t47d cell lines were cultured in dmem high glucose (invitrogen) with fetal bovine serum (fbs) 10% (gibco), penicilinstreptomycin 1.5% (gibco), and fungizone 0.5% (gibco). trypsin-edta 0.25% (gibco) was used to detach the cells from tissue culture dish. phosphate buffer saline (pbs) was used as washing solution. dmso (sigma) was used to dissolve the test solution in concentration less than 1%. for cytotoxicity assay, mtt (3-[4,5dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) reagent (sigma) 5 mg/ml was dissolved in dmem medium. sodium dodecyl sulfate (sds)-hcl 0.01 n was used as stopper reagent. for apoptosis assay, we used reagent kit from biovision. cytotoxic assay cells (briefly 6x10 3 cells/well) were transferred in to six well tissue culture plate (iwaki) and incubated for 24 h. cells were treated with fcb (concentration 20; 50; 75 and 100 µg/ml), cisplatin (concentration 1; 2; 5; 10; 15; and 30 µm), and their combination (concentration ratio 1/6; 1/3; and 1/2 ic50) and then incubated for 24 h. after 24 hours of incubation, medium was removed and cells were washed with 100 ml pbs. then 100 ml mtt reagent with final concentration 0.5 mg/ml in dmem high glucose medium was added into each well and incubated again for 34 hours to form formazan crystal. the stopper reagent (10% sds in 0.01 n hcl) was used to dissolved and incubated overnight at room temperature and in the dark (covered with aluminum foil). the next day, the absorbance from each well was measured by elisa reader with 595 nm wavelength. apoptosis assay cells (briefly 3x10 5 cells/well) were transferred in to six well tissue culture plate (iwaki) and incubated for 24 h. cells were treated with fcb, cisplatin, and their combination with concentration ½ ic50 and then incubated for 24 h. after incubation, adherent and detached cells were collected and centrifugated at 2000 rpm for 3 min, then washed twice with cold pbs. then, pbs was discarded, conical was sealed with aluminum foil, and 500 ml of buffer annexin-v were added to conical. annexinv and propidium iodide reagent were added respectively by 5 ml, incubate for 5 minutes and analyzed by using flowcytometer (bd, facs calibur). data analysis single cytotoxic assay. cell viability was determined by percent viability. the calculation is [(absorbance of treated-drug) (absorbance of medium)/(absorbance of control (untreated) cells absorbance of medium)] × 100%. linier regression between log concentration and % cell viability giving the equation y = bx + a were used to calculate ic50 value. the ic50 values are defined as the drug concentrations required to reduce the absorbance by 50% of the control, combinational cytotoxic assay, drug reduction inex (dri) and combination index (ci) was assessed by the chou-talalay method utilizing compusyn® software. result and discussion cytotoxic assay was performed using mtt assay. the results indicated the occurrence of dose dependent manner. ic50 value that obtained for fcb single treatment is 68 µg/ml (fig. 1). ic50 value that less than 100 µg/ml indicates that fcb has potent cytotoxic effect against t47d cells (omoyeni, et al., 2014). when compared with methanolic extract of caesalpinia sappan l., fcb also has more potent cytotoxic effect because the ic50 value from methanolic extract of caesalpinia sappan l. was 150.9 µg/ml (nurulita and muflih, 2006). single treatment of cisplatin against t47d breast cancer cells was also conducted to determine the ic50 value. the ic50 value of cisplatin that obtained in this study is 16 µm (fig. 2). tirtanirmala, et al., 2015 indones. j. cancer chemoprevent., 6(3), 89-96 91 figure 1. cytotoxic effect of fcb on t47d cells. t47d cells (6x103 cells/well) were seeded in 96 wellplate and treated with fcb 20, 50, 75, and 100 µg/ml. then plate was incubated for 24 hours and cells viability was determined by using mtt assay as described in methods. the higher concentration of fcb caused lower cells viability. data were mean of two replications x ± sd (p<0,05). ic50 value was calculated using linier regression and fcb perform cytotoxicity with ic50 value of 68 µg/ml. figure 2. cytotoxic effect of cisplatin on t47d cells. t47d cells (6x103 cells/well) were seeded in 96 wellplate and treated with cisplatin1, 2, 5, 10, 15, and 30 µm. then plate was incubated for 24 hours and cells viability was determined by using mtt assay as described in methods. the higher concentration of cisplatin caused lower cells viability. data were mean of two replications x ± sd (p<0,05). ic50 value was calculated using linier regression and cisplatin perform cytotoxicity with ic50 value of 16 µm. combination of cisplatin and fcb possessed synergistic effect in concentration ratio 1/6; 1/3; and 1/2 ic50 (table 1). the most synergistic effect was shown in concentration ratio 1/2 with ci value 0.66. dose reduction index (dri) value of fcb was 3.06, meant that fcb reduced the dose required when combined with cisplatin as much as 3.06 times (from 63.81 µg/ml to 20.85 µg/ml). it also happened with cisplatin that reduced the dose as much as 2.54 times (from 12.46 µm to 4.91 µm). dri was occured in 50% inhibition of viable cells. beside dri, combination index (ci) value also be analyzed from compusyn. by looking ci value, the combination of drugs can be classified to synergistic, additive or antagonistic based on interactions. from ci values, the combination between cisplatin with fcb had synergistic effect because its ci value was less than 1 (for concentration ratio 1/2 ic50, 1/3 ic50, and 1/6 ic50). the best ci value was occured when combined with concentration ratio 1/2 ic50 with ci value of 0.66 and decreased the percentage of cell viability up to 23%. cell morphology after treatment was observed. there were cell morphology change on treatment combination cisplatin and fcb with concentration ratio 1/4 and 1/2 ic50 (fig. 3b, 3c) compared with control cells (fig. 3a). 0 20 40 60 80 100 120 0 20 40 60 80 100 120 v ia b il it y c e ll ( % ) concentration (µg/ml) 0 20 40 60 80 100 120 0 10 20 30 40 v ia b il it y c e ll ( % ) concentration (µm) tirtanirmala, et al., 2015 indones. j. cancer chemoprevent., 6(3), 89-96 92 tabel 1. combination index (ci) and dose reduction index (dri) values for fcb, cisplatin, and their combination. data was obtained from compusyn software % sel hidup ci fraksi brazilein cisplatin konsentrasi (µg/ml) dri konsentrasi (µm) dri tunggal kombinasi tunggal kombinasi 95 1.02 30,30 5,26 5,76 1,47 1,24 1,18 90 0.90 36,60 7,46 4,91 2,52 1,76 1,44 75 0.78 48,33 12,47 3,87 6,61 2,94 1,91 50 0.72 63,81 20,85 3,06 12,46 4,91 2,54 30 0.71 79,06 31,00 2,55 23,07 7,29 3,16 figure 3. combinational cytotoxic effect of fcb and cisplatin on t47d cells. t47d cells (6x103 cells/well) were seeded in 96 wellplate and treated with fcb and cisplatin in concentration ratio ic50 : ic50 (1/2; 1/3; and 1/6 ic50). then, plate was incubated for 24 hours and cells viability was determined by using mtt assay as described in methods. combinational treatment altered cells morphology. cells treated with (a) control cells, (b) combination with concentration ratio ¼ ic50, and (c) combination with concentration ratio ½ ic50. white arrows ( ) showed viable cells while black arrows ( ) showed death cells were observed under light microscope with 100x magnification. graph (d) showed that combination of fcb and cisplatin decreased cells viability compared to cisplatin single treatment. 0 20 40 60 80 100 120 1 c e ll v ia b il it y ( % ) cell control fcb 50 µg/ml cisplatin 10 µm combination (1/3 ic50) combination (1/2 ic50) (a) (b) (c) (d) * * tirtanirmala, et al., 2015 indones. j. cancer chemoprevent., 6(3), 89-96 93 cytotoxic activity of compound was correlated to its ability to induce apoptosis or inhibit cell cycle. apoptosis assays’ result (fig. 4) showed that almost 90% of control cell population was not observed the presence of cell death. while on fcb treatment, apoptosis induction was observed as much as 80.59% of cell population. on cisplatin treatment, apoptosis induction observed by 80.86% of cell population. meanwhile, the percentage of apoptotic cells in combination treatment was 93.93%. it proved that combination treatment of fcb and cisplatin with 24 hour incubation enhance the ability of cisplatin to induced apoptosis as much as 13% on t47d breast cancer cells. figure 4. effect of combination fcb-cisplatin on t47d cells apoptosis. t47d cells were seeded at 3x105 cells/well on 6 wells tissue culture plate, then treated with fcb ½ ic50 alone and its combination with cisplatin ½ ic50. after 24 hours of incubation, cells were harvested as described in methods, added with annexinv and pi reagent, then subjected to facs flowcytometry. flowcytometric profile of cells treated with (a) control, (b) fcb, (c) cisplatin, and (d) combination of fcb and cisplatin. there are 4 quadrans: lower left (ll) indicates viable cells, lower right (lr) indicating early apoptotic cells, upper left (ul) indicating late apoptotic cell, upper right (ur) indicating necrotic cells. graph (e) showed the percentage of viability cell of combination fcb and cisplatin induce apoptosis. 0 10 20 30 40 50 60 70 80 90 100 p e rc e n ta g e ( % ) cell control fcb cisplatin combination sample id: ks t47d.2 patie nt id: 040 7.14 acqu isition date: 07-apr-14 gate: no g ate tota l even ts: 20000 quad location : 32, 30 quad % gated % total ul 0.67 0.67 ur 4.15 4.15 ll 87.06 87.06 lr 8.12 8.12 (a) sample id: fb t47d patie nt id: 040 7.14 acqu isition date: 07-apr-14 gate: no g ate tota l even ts: 20000 quad location : 32, 30 quad % gated % total ul 5.76 5.76 ur 6.48 6.48 ll 13.64 13.64 lr 74.11 74.11 (b) sample id: cisp t47 d patie nt id: 040 7.14 acqu isition date: 07-apr-14 gate: no g ate tota l even ts: 20000 quad location : 32, 30 quad % gated % total ul 13.52 13.52 ur 23.94 23.94 ll 5.62 5.62 lr 56.92 56.92 (c) sample id: fb cisp t4 7d patie nt id: 040 7.14 acqu isition date: 07-apr-14 gate: no g ate tota l even ts: 20000 quad location : 32, 30 quad % gated % total ul 0.90 0.90 ur 5.37 5.37 ll 5.17 5.17 lr 88.56 88.56 (d) (e) tirtanirmala, et al., 2015 indones. j. cancer chemoprevent., 6(3), 89-96 94 t47d breast cancer cell lines expreses caspase-3 wildtype, caspase-7 wildtype, mutant p53 and er/pr-positive (bouker, et al., 2005; schafer, et al., 2000). in fact, p53 tumor suppressor gene is a gene that plays a role in apoptosis. this p53 gene stimulates expression of bcl-2 family including bax, and can bind to one or more anti-apoptotic proteins in mitochondria, such as bcl-xl (chipuk, et al., 2003; marsden, et al., 2002). on the other hand, cisplatin also plays a role in mediating the activation of the p53 protein, which is followed by the occurrence of dna damage (tanida, et al., 2012), so the mutation of p53 gene on t47d cells can inhibit cisplatin to induce apoptosis. however, previous studies had proved that the mechanism of cisplatin to induce apoptosis was by causing downregulation of bcl-2 gene on mcf 7 cell lines (thomadaki and scorilas 2007) and t47d cell lines (mokhtari, et al., 2012). other studies had also demonstrated the expression of bcl-2, bclxl, bad and bax was regulated by cisplatin (siervo-sassi, et al., 2003). so, that is the reason of cisplatin still can induce apoptosis on t47d cell lines, which is expresses mutant p53. cytotoxic activity of fcb on t47d cells was expected to be caused by active compounds contained in these fractions, namely brazilin and brazilein. cytotoxic mechanism of combination fcb and cisplatin occured through apoptosis induction. apoptosis induction of fcb alone might be related to the mechanism of apoptosis induction from p53 independent. brazilin which is the main compound from secang’s heartwood can activate caspase-3 and regulate the expression of bcl-2 family proteins, including bax, bcl-x (l), and bcl-2 on u 266 myeloma cancer cells (kim, et al., 2012). brazilin also known could inhibit proliferation of glioblastoma cells (yen, et al., 2010) and induce apoptosis on glioblastoma cells through activation of caspase-3, followed by poly-(adp)-ribose polymerase (parp) cleavage and through the induction of caspase 7 (lee, et al., 2013; hengartner, 2000). in addition, brazilin also increased the population of glioblastoma cells in cell cycle sub-g1 area (lee, et al., 2013). while the other major compound of secang’s heartwood, like brazilein, also known could increase the activation of caspase-9 and caspase-3, induce parp cleavage and cause down-regulation of survivin protein on hepg2 liver cancer cells (zhong, et al., 2009). on mcf-7 breast cancer cells, brazilein also could induce apoptosis through inhibition of survivin expression (tao, et al., 2011). brazilein also 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synthesis and evaluation of brazilein and analogs as antiinflammatory and cytotoxic agents, bioorg. med. chem. lett., 20(3), 10371039. zhong, x., wu, b., pan, y.j. and zheng, s., 2009, brazilein inhibits survivin protein and mrna expression and induces apoptosis in hepatocellular carcinoma hepg2 cells, neoplasma, 56(5), 387-92.  95 indonesian journal of cancer chemoprevention, june 2019 issn: 2088–0197 e-issn: 2355-8989 selectivity index of alpinia galanga extract and 1’-acetoxychavicol acetate on cancer cell lines muhammad da’i1,*, khairunnisa azani meilinasary1, andi suhendi1, sari haryanti2 1pharmaceutical chemistry department, faculty of pharmacy, universitas muhammadiyah surakarta, surakarta, indonesia 2the center for research and development of traditional medicinal plants and medicines (b2p2toot), karanganyar, indonesia abstract previous research stated that galangal (alpinia galanga) extract has a potential as cytotoxic agent with active compound of 1’-acetoxychavicol acetate (aca). the objective of this study was to determine the selectivity of ethanol extract, ethyl acetate fraction, and methanol fraction of of galangal, and aca on cancer cell lines. cytotoxic activity was carried out using the mtt method on t47d breast cancer, widr colon cancer, hela cervical cancer, and vero normal cell lines. the results showed that galangal ethanol extract and its fractions had selectivity index equal to or less than 2 on cancer cells. meanwhile, aca had selectivity index more than 3 on t47d cell and hela cell. aca showed a strong cytotoxic activity against cancer cells t47d, hela, and widr with ic 50 values of 3.14, 7.26, and 12.49 μg/ml, respectively. based on data, it could be concluded that aca was the most selective to inhibit t47d cell with a selectivity index of 6.6. keywords: 1’-acetoxychavicol acetate, galangal (alpinia galanga), selective index, cytotoxic introduction cancer is a disease that causes high mortality in the world. in 2012, 8.2 million deaths have been caused by this disease (pusat data dan informasi, 2015). chemotherapy, surgery, radiation, and hormonal therapy are the number of common therapies for cancer patients; however, they require high costs as well as a number of side effects due to the low selectivity of therapy. researchers therefore continue to conduct research to obtain more selective anticancer drugs. one of the potential ingredients to be developed as anti-cancer with better selectivity is herbal medicines. one source of herbal medicines that has been traditionally used for curing cancer is galangal (alpinia galanga) (kuntorini, 2005). zaeoung, et al., (2005) stated that alpinia galanga as an antioxidant and free radicals scavenger has cytotoxic activity against mcf7 (breast adeno-carcinoma) and ls174t (intestinal adenocarcinoma) cells. galangal extract at the dose of 225, 450, and 750 mg/kgbw/day could increase the apoptosis process and reduce proliferative activity in breast cancer cells (hartono, 2009). cytotoxic submitted: june 17, 2019 revised: june 27, 2019 accepted: june 27, 2019 *corresponding author: muhammad.dai@ums.ac.id 96 da’i, et al., 2019 indones. j. cancer chemoprevent., 10(2), 95-100 activity, described as ic50 value, on hela cervical cancer cell line of galangal extract from three local markets were 13.26, 36.32, and >100 µg/ml in order. meanwhile, alpinia galangal extract (age) from pasar legi (surakarta, indonesia) on mcf7 and t47d breast cancer cell lines have ic50 value of 15.80 and 12.50 µg/ml, respectively (suhendi et al., 2017). galangal contains several phenylpropanoid compounds, including 1'-acetoxychavicol acetate (aca), 1'-acetoxyeugenol acetate, trans-pcoumaril diacetate, 1'hydroxyccapsol acetate, and trans-coumaryl alcohol (matsuda, et al., 2005). aca is the main composition of alpinia galanga (baradwaj, et al., 2017; hasima, et al., 2010). aca has cytotoxic against various cancer cell lines such as a549 cancer cells (lung cancer), snu638 (stomach cancer), hct116 (colon cancer), ht1080 (fibrosarcoma), and hl60 (leukemia) with ic50 values of 8.14, 1.27, 1.77, 1.20, and 2.39μg/ml, respectively (nam, et al., 2005). another research by zeng, et al., (2015) revealed that aca showed cytotoxic activity on hela (cervical cancer), a549 (lung cancer), hepg-2 (liver cancer) and smmc-7721 (liver cancer) with ic50 values of 85.1, 64.44, 74.51, and 61.27μg/ml, respectively. this study was conducted to determine the selectivity of galangal extract and aca in breast cancer cells (t47d), cervical cancer cells (hela) and colon cancer cells (widr) compared to normal cells (vero). the finding would serve as a basic for the further development targeted on cytotoxic research. method materials that were used in this study include evaporator (heidolph), waterbath (changzhou nuohai xmtd-204), analytical balance (sartorius), micro pipette (soccorex), laf (nuaire), hemocytometer (marienfield germany), 96-well-plate (iwaki), conical tube, elisa reader (biotek), incubator (binder), microscope (olympus), galangal rhizome (laboratorium balai besar penelitian dan pengembangan tanaman obat dan obat tradisional, tawangmangu, indonesia ), acetoxychavicol acetate (lkt laboratories inc.), roswell park memorial institute (rpmi 1640, gibco), dimethyl sulfoxide (dmso) (merck), fetal bovine serum 10% (fbs, gibco), penicillin-streptomycin 1% (gibco), tripsin (gibco), sodium dodecyl sulfate (sds, gibco), mtt (sigma), fungizone (gibco). cell lines (t47d, hela, and widr) were obtained from laboratorium balai besar penelitian dan pengembangan tanaman obat dan obat tradisional tawangmangu, karanganyar, indonesia (b2p2toot). extraction and fractionation extraction was conducted by maceration process with ethanol 95% as solvent within 3 days. liquid extract was then evaporated to obtain the thick extract. ten milligrams of thick extract were dissolved in 10 ml of distilled water and 10 ml of ethyl acetate in a separating funnel. the top layer (ethyl acetate partition) was taken and evaporated in a water bath covered in aluminum foil to form the ethyl acetate fraction. subsequently, the ethyl acetate fraction was dissolved using methanol (methanol fraction). the fractionation was done in triplicates. mtt assay the mtt reagent (0.5 mg/ml) was prepared by taking 1 ml of stock solution of mtt in pbs (50 mg/10 ml) and diluted it by media up to 10 ml (for 1 well plate). once disposed, the cell was washed with pbs and 100 µl mtt reagent was added to each well, including media control (without cells). the cell, following this, was incubated for 2-4 hours in a co2 incubator. the cell was then examined with an inverted microscope. after formazan was clearly formed, a stopper reagent (sds 10%) was added in 0.1 n hcl. the plate was wrapped with paper or aluminum foil and incubated in a dark place at room temperature for one night. absorbance of each well was then read by an elisa reader at λ = 595 nm. ic50 was calculated based on linier regression equation between viability cells and concentration of samples (mosmann,1983).  97 indonesian journal of cancer chemoprevention, june 2019 issn: 2088–0197 e-issn: 2355-8989 selectivity index analysis selectivity index (si) is obtained from the ic50 value of a compound against normal cells divided by the ic50 value of cancer cells (aljewari, et al., 2010; badisa, et al., 2006). compounds are classified as high selectivity if the si value is >3 and less selective if the si value is <3 (sutejo, et al., 2016). results and discussion the material used in this study is galangal rhizomes obtained from the center for research and development of traditional medicinal plants and medicines (b2p2toot) tawangmangu, karanganyar regency, central java. morphologically, the fresh galangal rhizomes are characterized with small and thick, fleshy, cylindrical about 2-4 cm in diameter, and branched. the outer parts are rather brown, reddish or pale greenish yellow with white and reddish, hard glossy scales, and while the inside part is white. the flesh of old rhizomes is rough fibrous. when getting dried, the rhizomes turn somewhat greenish, and the fibers become hard and tough. dried rhizome was then extracted by maceration using 96% ethanol as solvent. the result of thick extract was 73.09 g with yield of 8.12%. to group chemical constituents in extract, fractionation was conducted. the method used in fractionation of galangal extract was a liquid-liquid extraction. grouping chemical constituent was based on distribution constanta of chemicals in non-immiscible solvent (berthod and carda-broch, 2004). the target of fractionation was to obtain the rich fraction of active constituent. one of active compounds of galangal extract is aca, which has potential as an anticancer (asri and winarko, 2016). fractionation results showed the yield of ethyl acetate fraction obtained as 7.43 gram, higher than the methanol fraction as 1.30 gram. the cytotoxicity determination of galangal ethanol extract, ethyl acetate fraction, methanol fraction and aca compounds were carried out using the mtt method on breast cancer cells (t47d), cervical cancer cells (hela), colon cancer cells (widr), and normal cells (vero). morphology of cancer cells after treated by samples is shown in figure 1. the value of ic50 of samples were calculated based on regression equation (figure 2) of % viability cell percentage vs concentration. the value of ic50 of samples on three cancer cells showed a strong cytotoxic activity (table 1). due to ic50 values, the aca is viewed to have the most active cytotoxic activity against all cancer cells tested. ethyl acetate fraction has better cytotoxic activity compared to ethanol extract and methanol fraction. as aca compounds are semi-polar, they could dissolve more in ethyl acetate. the compounds have the most active cytotoxic activity in t47d cells followed by hela cells and widr cells. ic50 values of vero t47d hela widr figure 1. morphology of cancer cells after administration of aca at a dose of 10μg/ml. the cells were observed after 24 h of treatments under an inverted microscope with magnification of 100x. 98 da’i, et al., 2019 indones. j. cancer chemoprevent., 10(2), 95-100 aca in t47d, hela, and widr were found in 3.14; 7.26, and 12.49μg/ml, respectively. the potential of a compound is classified as strong cytotoxic agent if the ic50 value <20µg/ml, moderate if the table 1. selectivity index of extract, ethyl acetate, methanol fractions and aca on t47d, hela, widr, and vero cell lines. ic50 (μg/ml) si ic50 (μg/ml) si ic50 (μg/ml) si ic50 (μg/ml) si ethanol extract 44.93 ± 1.08 1.6 40.00 ± 0.55 1.9 66.11 ± 3.15 1.1 74.06 ± 7.13 1 ethyl acetate fraction 42.29 ± 3.28 1.3 35.54 ± 0.44 1.5 48.81 ± 1.68 1.1 53.66 ± 2.57 1 methanol fraction 40.49 ± 2.13 1.8 35.76 ± 1.53 2 55.24 ± 1.62 1.3 72.77 ± 0.35 1 aca 3.14 ± 0.14 6.6 7.26 ± 0.12 3.5 12.49 ± 1.09 2 25.25 ± 0.92 1 samples t47d hela widr vero a b c d figure 2. graphics of correlation of viability cell percentage vs concentration of aca, ethanol extract of alpinia galangal, and its fractions. percentages of viable cells were obtained based on colorimetric reaction of mtt that reduced by reductase enzyme resulting formazan and the absorbance was measured on wavelength 550 nm. ethanolic extract showed negative slope that indicated the cytitoxic effect and aca most potent to inhibit the cell growth on vero, t47d, hela, and widr cell lines. a: ethanol extract of alpinia galangal; b: methanol fraction alpinia galangal; c: ethyl acetate fraction alpinia galangal; d: 1’-acetoxychavicol acetate (aca). ic50 value <50 μg/ml, and iweak if the ic50 value > 50μg/ml (ellithey, et al., 2014) (table 1). from the results obtained, it can be concluded that aca has strong cytotoxic properties.  99 indonesian journal of cancer chemoprevention, june 2019 issn: 2088–0197 e-issn: 2355-8989 the selectivity of chemopreventive agents means that only cells identified as cancer cells are attacked. the higher the selectivity index of a compound to cells, the more selective the compounds to kill or inhibit the growth of a cancer cell with the smaller effect on normal cells. the small selectivity index indicates that the compound is less toxic to normal cells rather than to cancer cells. compounds with high si values offer the potential for safer and more effective therapy in cancer therapy (segun, et al., 2019). based on the test results galangal ethanol extract, ethyl acetate fraction, and methanol fraction are found to be less selective for all tested cells (si<3). meanwhile, aca is selective for t47d and hela cells with si>3 and less selective for widr cells. the results of this research need a further study to determine the anticancer potential in animal models. due to the lack of selectivity and many side effects (such as fatigue, nausea, alopecia and others) of cancer chemotherapy, these finding promising to develop as selective anticancer agent (gonzález-arriagada, et al., 2013; ihbe-heffinger, et al., 2013). conclusion the extract and fractions of alpinia galangal ethanol extract have potential cytotoxic activity on t47d, hela and widr. compound 1’-acetoxychavicol acetate has a strong cytotoxic activity against cancer cells t47d, hela and widr with ic50 values of 3.14, 7.26 and 12.49μg/ml, respectively and selective for t47d breast cancer cells with selectivity index of 6.6. acknowledgment this work has been funded by universitas muhammadiyah surakarta by pinpru research scheme. references aljewari, h., al-faisal, a.h.m. and nader, m., 2010, in vitro cytotoxic activity of the l-asparaginase extracted and purified from pathogenic escherichia coli against four leukemic cell lines. presented at the 2nd annual international conference of northest pharmacy research, faculty of pharmacy, mahasarkham university, thailand, pp. 21–23. asri, a. and winarko, s., 2016, antiproliferative activity by ethanolic extract of red alpinia 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inducing cancer cell death from the seeds of alpinia galanga, a chinese spice, food funct., 6, 431–443. indonesian journal of cancer chemoprevention, june 2017 issn: 2088–0197 e-issn: 2355-8989 61 doxorubicin induces lamellipodia formation and cell migration nur dina amalina 1 , ika putri nurhayati 1,3 , edy meiyanto 1,2* 1 cancer chemoprevention research center (ccrc), faculty og pharmacy, universitas gadjah mada, yogyakarta 2 department of pharmaceutical chemistry, faculty of pharmacy, universitas gadjah mada, yogyakarta 3 department of pharmacy, faculty of medicine, universitas brawijaya, malang abstract breast cancer is the main cause of cancer death among women, especially breast cancer metastasis. metastasis process begins with the ability of cell cancer invasion. doxorubicin, a antracycline chemotheraphy, is known to induce tgfβ1, thus promote invasion. the aim of this study is to optimize doxorubicin doses to induce lamellipodia formation in 4t1 and mcf-7/her2 cells. lamellipodia formation was observed by morphological changes using microscope inverted. the effect of doxorubicin on cell viability was analyzed using mtt assay. rac1 expression after doxorubicin exposure was determined by western blotting. lamellipodia formation was observed by morphological change of the cell at the dose 10, 25, 50 and 100 nm. doxorubicin at the dose of 10 nm could induced lamellipodia formation without affect cell viability in both 4t1 and mcf7/her2 cells. doxorubicin induced cell cycle arrest at g2/m phase at all doses. doxorubicin 10 nm also decrease rac1 expression compared to control. keywords: doxorubicin, lamellipodia, rac1, migration. introduction metastasis, one of hallmark of cancer, is spreading of cancer cell from primary site to distant site. therapy of cancer metastasis becomes challenging and metastasis is the main cause of cancer death among patient. metastasis process begin with local invasion, cells detached from primary site, increase motility and release proteolytic enzyme to degrade extracellular matrix, such as serine, cysteine, and metalloprotease (verma and hansch, 2007). metastasis was characterized by loss of e-cadherin, a molecule involved in cell adhesion (hanahan and weinberg, 2011). cell migration depend on cell polarization and lamellipodia formation via rac1 cascade. rac1 activation induce actin polymerization and increase cell motility (arpaia, et al., 2012). doxorubicin is widely used as chemotherapy in breast cancer. long-term use of doxorubicin cause some side effects leading to reduce chemotherapy effectivity. in addition, doxorubicin also induced tgfβ1 signaling activity in triple negative receptor breast cancer cell type and mda-mb 231. doxorubicin induced epithelial-mesenchymal transition (emt) and initiate invasion via tgfβ1 signaling. thus, doxorubicin could induce cell migration to distant site. on the other hand, activation of tgfβ1 signaling also generate stemlike breast cancer cell and increase drug resistance (bandyopadhyay, et al., 2010). the aim of this study is to optimize doxorubicin doses to induce lamellipodia formation on 4t1 and mcf-7/her2 cells. methods cell culture breast cancer cell line culture type 4t1 (originally from atcc r -crl-2539 tm ) and mcf7/her2 were obtained from prof. masashi kawaichi (nara institute of science and technology, naist, japan). the cells were maintained in dulbecco’s modifies eagles medium (dmem) high glucose (sigma) supplemented with 10% fbs (sigma), hepes, sodium bicarbonate, 1.5% penicilin-streptomycin and 0.5% fungizone (gibco). cells were cultured with 5% co2 in 37 o c. doxorubicin sample preparation doxorubicin was dissolved in dmso (sigma), and freshly diluted in culture medium in several concentrations before used. *corresponding author e-mail : edy_meiyanto@ugm.ac.id amalina, et al., 2017 indones. j. cancer chemoprevent., 8(2), 61-66 62 morphologycal lamellipodia formation murine 4t1 cells were treated with doxorubicin (10, 25, 50, and 100 nm) for 96 hours. the medium with doxorubicin was changed every alternate day. representative pictures of lamellipodia formation detected by inverted microscopy (x100 magnification) (bandyopadhyay, et al., 2010). proliferation assay approximately 2x10 3 4t1 cells/well and mcf-7/her2 2.5x10 3 4t1 cells/well were seeded in 96-well plates and incubated for 24 hours. cells were treated with increasing concentration of doxorubicin (10, 25, 20, and 100 nm) for 24, 48, and 72 hours. cultured medium was removed and cells were washed with pbs (sigma). mtt 0.5 mg/ml in medium were added into each well and incubated for 3-4 hours. mtt reaction was stopped by the addition of sds 10% in hcl 0.01 n, and incubated overnight in the dark room. the absorbance was measured using elisa reader at λ 595 nm (merck elisa reader). each treatment were carried out in triplicate, and the absorbance data were provided as percent viability compared to control cells (untreated) (mosmann, 1983). cell cycle analysis the 4t1 cells (7.5x10 4 cells) were seeded in 6-well plate and incubated for 24 hours. cell were treated with doxorubicin 10, 25, 50 and 100 nm for 24 hours after the treatment, cells were harvested by trypsinization and stained with the staining solution contains propidium iodide (pi) 1 mg/ml protease inhibitor, 10 mg/ml rnase and 0,1% (v/v) triton x-100 (merck). cells were incubated for 5 minutes in the dark room, transferred into a flow cytometric tube and analyzed by bd facs calibur (bd bioscience, usa). cell cycle distribution was acquired by using flowing software. western blot approximately 4x10 3 4t1 cells and 3x10 3 mcf-7/her2 cells were seeded in 6-well plates, and incubated for 24 hours. cell were treated with doxorubicin 10, 25, 50 and 100 nm for 48 hours. protein was extracted using radio immune precipitation assay (ripa) buffer (tris hclph 7.6, np 40, na-deoxycholate, nacl, sds, phenyl methyl sulfonyl flouride (pmsf), naf, and cocktail inhibitor protease, then separated in 14% acrylamide gel by sds-page electrophoresis. after transferring to polyvinylidene fluoride (pvdf) membrane, the membrane was incubated overnight at 4°c with either the mouse monoclonal antibody against rac1 (santa cruz cs22475) or β-actin (santa cruz sc47778). after incubation with secondary antibody anti-mouse (santa cruz sc-516102) for 1 hour, the protein bands were visualized using ecl (amersham) and detected using luminograph. the relative protein levels were calculated in reference to the amount of β-actin protein. results the effect of doxorubicin in cells proliferation the cytotoxic drug doxorubicin is a wellknown chemotherapeutic agent which is used in treatment of a wide variety of cancers inducing intracellular ros accumulation, cell cycle arrest and apoptosis. however, doxorubicin treatment is often hampered by severe side effects and resistance of cancer cells to therapy. to determine the effect of doxorubicin in inducting lamellipodia formation on 4t1 and mcf-7/her2 cells, firstly we examined the cytotoxic effect of doxorubicin at different concentrations (0, 10, 25, 50 and 100 nm) for 0, 24, 48 and 72 hours by mtt assay. as shown in fig. 1, doxorubicin demonstrated a cytotoxic effect on 4t1 and mcf-7/her2 cells. doxorubicin reduced the viability on both of cells in a dose-dependent and a time-dependent manner. effect of doxorubicin on cell cycle modulation in order to investigate the mechanism of physicological changes focusing on cell cycle, the cells were processed for flowcytometry by pi staining. our study showed that cell proliferation was affected by doxorubicin treatment with concentration dependent manner. furthermore, cell cycle analysis was elaborated to understand the modulation of cell proliferation through cell cycle alteration. here, we found that doxorubicin treatment conducted cell to perform g2/m arrest and increase accumulation of sub-g1 phase cells (fig. 2). the highest effect of cell cycle alteration showed in 25 nm doxorubicin treatment in both phase. interestingly, the cell percentage of each phase was different. whereas, the 10 nm of doxorubicin gave similar proliferation effect, similar to the control. hence, it needed to explore further about the morphological alteration due to different concentration of doxorubicin. amalina, et al., 2017 indones. j. cancer chemoprevent., 8(2), 61-66 63 figure 1. proliferation effect of doxorubicin (doxo) on cell viability. a. proliferative assay of doxo 10, 25, 50, and 100 nm on 4t1 cells. b. proliferative assay of doxo 10, 25, 50, and 100 nm on mcf-7/her2 cells. proliferative assay was determined by mtt assay as described in the methods. cell was treated with various concentration of doxo for 24, 48, and 72 hours. cell viability after treatment was compared to baseline. each experiment was conducted in triplicate. mean values from the three experiments ± standard error of mean are shown. c e ll n u m b e r dna content i ii iii iv a b 4.22 7.46 7.99 6.23 43.70 14.21 4.78 1.24 27.55 18.65 8.40 19.44 24.52 59.68 78.84 73.09 0 20 40 60 80 100 control doxo 10 nm doxo 25 nm doxo 100 nm c e ll n u m b e r (% ) g2/m s g1 sub g0/g1 figure 2. effects of doxorubicin on cell cycle modulation on 4t1 cells. cells were harvested after 24 h of treatment, stained with propidium iodide and dna content were analyzed by using flowcytometry. (a) the profiles of cells in the phases of sub-g1, g1, s and g2-m (i) control cells, (ii) doxorubicin 10 nm, (iii) doxorubicin 25 nm, (iv) doxorubicin 100 nm. x-axis showed the relative content of dna and the y axis showed the relative cell numbers. (b) quantification of cells distribution in each phase (sub g0/g1, g1, s, and g2/m phase) of the various treatments. the percentage of sub-g1 phase cells showed apoptotic cell population. effect of doxorubicin on lamellipodia formation lamellipodia formation is beginning step of cell migration, thus effect of doxorubicin on lamellipodia formation was observed. the cells were treated using doxorubicin at the dose of 10, 25, 50, and 100 nm. lamellipodia formation was observed at 0, 24, 48, 72, and 96 hours after treatment (fig. 3). change of cell morphology also observed. lamellipodia of 4t1 cells was observed even at lowest doses (10 nm) after 24 hours treatment. meanwhile lamellipodia of mcf-7/her2 cells was observed at the doses of 25 nm after 24 hours treatment. prolong exposure of doxorubicin on both cells after 96 hours treatment affect cell morphology leading to cell death. effect of doxorubicin on rac1 expression rac1 is protein involved in rearrangemnet of actin and lamellipodia formation. to understanding whether lamellipodia formation were affected by rac1 expression, thus rac1 expression was observed. rac1 expressions of 4t1 and mcf7/her2 after treatment with doxorubicin 10 nm was examined using western blot. doxorubicin 10 nm reduced rac1 expression in both 4t1 and mcf7/her2 cells (fig. 4). due to this result, we understood that the formation of lamellipodia was different on both cell yet the treatment of 10 nm doxorubicin brought similar effect on rac1 expression. amalina, et al., 2017 indones. j. cancer chemoprevent., 8(2), 61-66 64 figure 3. induction of lamellipodia formation by doxorubicin (dox). (a) on 4t1 cells, (b) on mcf-7/her2 cells. representative pictures of 4t1 cells with mesenchymal morphology after treatment with untreated cells, dox 10 nm, dox 25 nm, dox 50 nm and dox 100 nm. shown is confocal microscopy of the lamellipodia (100x magnification). arrows indicate cells morphological changes. a b amalina, et al., 2017 indones. j. cancer chemoprevent., 8(2), 61-66 65 figure 4. rac-1 protein expression under doxorubicin (doxo). (a) 4t1 cell and (b) mcf-7/her2 after treatment with doxo 10 nm. expression was compared between control (untreated) and doxo treatment. rac1 expression was observed with western blot shown different expression between groups. the levels of expression were normalized by comparing with β-actin expression level discussion doxorubicin is usually for first-line treatment of several type cancer. drug chemosensitivity have been investigated in the several models of breast cancer cell such as t47d, widr colon cancer cells, mcf-7, mcf-7/dox and showed various cytotoxic effects (febriansah, et al., 2014; hermawan, et al., 2010; putri, et al., 2016). this study explored the effect of doxorubicin on triple negative breast cancer cell, 4t1, that is highly metastasis in breast and her2-overexpresed cell, mcf-7/her2. the results of our cytotoxicity assay, doxorubicin inhibited cell growth in a dose-dependent and a time-dependent manner on 4t1 and mcf-7/her2. previous study reported the cytotoxic mechanism of doxorubicin was through disruptions topoisomerase ii, resulting in dna damage and cell death (rowan, 1979). based on the result of cytotoxic effect of doxorubicin. this present study was continued to investigate possibility pathway in growth cells inhibition through cell cycle modulation. this study showed that doxorubicin inhibited 4t1 breast cancer cell growth through cell cycle modulation. three concentration of doxorubicin caused cell accumulation in g2/m phase. similar results were performed by previous studies that had been reported putri, et al (2017) which showed doxorubicin could inhibit the growth of t47d cells by an accumulation of cells at the g2/m phase. in this present study inhibition of cancer metastasis after treatment of doxorubicin was also observed. cell migration is part of the process of metastasis. interestingly, prolong use of doxorubicin exhibit several limitation such as toxicity and cancer cell resistance. on the other hand, doxorubicin activates tgf-β signalling in breast cancer cells which is increasing of tgf-β involved in mallignancies, such as metastatic breast cancer progression and survival. these also induce cancer cell migration and invation. activation of tgf-β also induce ephitelial-mesencymal transition that increase cell motility and invasiveness (bandyopadhyay, et al., 2010). the ability of cell to migrate involved formation of lamellipodia. the result of this study showed that low doses of doxorubicin could induced lamellipodia formation. doxorubicin at low doses induced lamellipodia formation both in 4t1 and mcf-7/her2 cell observed begin at 24 hours after treatment. this phenomenon was caused by doxorubicin ability to activate tgf-β leading to activation of smad2 and smad3. increasing of smad2 and smad3 induce emt, thus enhanced cell migration. in addition, activation of erk map kinase, rho gtpase and pi3 kinase/akt pathway followed by tgf-β also induced cell growth, migration, and invasion (xu, et al., 2009). extension of lamellipodia was needed by cell to move on certain direction. thus, observation of lamellipodia formation can be used as parameter to observed cell migration. rac1 is family of rho gtpase protein that involved in cellular dynamic, particularly lamellipodia that initiate migration of cell (ehrlich, amalina, et al., 2017 indones. j. cancer chemoprevent., 8(2), 61-66 66 et al., 2002). nevertheless, rac1 is not the only protein that regulate actin structures, rhog or cdc42 also play role in these structure (steffen, et al., 2013). in this study, doxorubicin reduce rac1 expression of 4t1 and mcf-7/her2 cells. these result indicate that lamellipodia formation on those cell did not dependent to rac1 expression. rab5, a member of rab family of gtpase, triggered lamellipodia formation independent with receptor tyrosine kinase/ras/pi3-k/rac pathways (spaargaren and bos, 1999). however, further study need to be conducted to confirm whether this phenomenon involved rab5 activity. conclusion doxorubicin could inhibits cells growth by induced g2/m cell cycle arrest and the other hand doxorubicin induce epithelial mesenchymal transition through rac1 independent-lamellipodia formation. acknowledgement this research was financially supported by research grant from the ministry of research, technology and higher education of the republic of indonesia. references arpaia, e., blaser, h., quintela-fandino, m., duncan, g., leong, h.s., ablack, a., et al., 2012, the interaction between caveolin-1 and rhogtpases promotes metastasis by controlling the expression of alpha5integrin and the activation of src, ras and erk, oncogene, 31(7), 884-896. bandyopadhyay, a., wang, l., agyin, j., tang, y., lin, s., yeh, i.-t., et al., 2010, doxorubicin in combination with a small tgfβ inhibitor: a potential novel therapy for metastatic breast cancer in mouse models, plos one, 5(4), e10365. ehrlich, j.s., hansen, m.d.h. and nelson, w.j., 2002. spatio-temporal regulation of rac1 during localization and lamellipodia dynamics epithelial cell-cell adhesion, dev. cell, 3(2), 259–270. febriansah, r., dyaningtyas, d.p.p., sarmoko, nurulita, n.a., meiyanto, e. and nugroho, a.e., 2014, hesperidin as a preventive resistance agent in mcf–7 breast cancer cells line resistance to doxorubicin, asian pac. j. trop. biomed., 4(3), 228–233. hermawan, a., meiyanto, e. and susidarti, r.., 2010, hesperidine increases cytotoxic effect of doxorubicin on mcf-7 cells, indones. j. pharm., 21(1), 8–17. hermawan, a., nur, k.a., sarmoko, dewi, d., putri, p. and meiyanto, e., 2012, ehanolic extract of moringa oleifera increased cytotoxic effect of doxorubicin on hela cancer cells, journal of natural remedies, 12(2), 108-114. mosmann, t., 1983, rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, j. immunol. methods., 65(1-2), 55–63. putri, h., jenie, r.i., handayani, s., kastian, r.f. and meiyanto, e., 2016, combination of potassium pentagamavunon-0 and doxorubicin induces apoptosis and cell cycle arrest and inhibits metastasis in breast cancer cells, asian pac. j. cancer prev., 17(5), 2683–2688. rowan t. chlebowski., 1979, adriamycin (doxorubicin) cardiotoxicity: a review, west j. med., 131(5), 364-368. spaargaren, m. and bos, j.l., 1999, rab5 induces rac-independent lamellipodia formation and ccell migration, mol. biol. cell, 10(10), 3239–3250. steffen, a., ladwein, m., dimchev, g.a., hein, a., schwenkmezger, l., arens, s., et al., 2013, rac function is crucial for cell migration but is not required for spreading and focal adhesion formation, j. cell sci., 126(20),4572-4588. xu, j., lamouille, s. and derynck, r., 2009, tgf-βinduced epithelial to mesenchymal transition, cell res., 19, 156–172. estrogenic effects of banana peels (musa paradisiaca l indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 151 banana peels (musa paradisiaca l.) extract as phytoestrogen on ovariectomized mice mammary gland development by inducing c-myc expression nanda resa pratama, yurista gilang, rita riata, adam hermawan, muthi’ ikawati, edy meiyanto *) cancer chemoprevention research center (ccrc) faculty of pharmacy universitas gadjah mada yogyakarta abstract hormone replacement therapy (hrt) is therapy for estrogen deficiency and post menopausal syndromes, but high cost and unwell-secured therapy. one of alternative therapy is the usage of phytoestrogens. the banana peel contains flavones, flavonol, flavanone and polimethoxyflavone which are potential as phytoestrogen. the purpose of this study was to examine the estrogenic effect of banana peel extract (bpe) development of mammary gland of ovariectomized rats. estrogenic effects was examined based on in vivo and in silico experiment. for in vivo experiment, female sprague-dawley rats aged 50 days were ovariectomized. at 70 days of age, 12 rats were treated with bpe 500 mg/kgbb and 1000mg/kgbb, 5 rats were treated with estradiol 2μg/day while others served as control were treated with cmc-na 0.5% and sacrificed 2 weeks later. the base line ovariectomized rats and base line non-ovariectomized rats were sacrificed at 70 days of age. the in silico experiment examined by molecular docking between myricetin and estrogen receptor alpha (er-α). the result of in vivo experiment showed that 1000 mg/kgbw bpe induced c-myc expression and enhance ovariectomized rat mammary gland development significantly. meanwhile, molecular docking showed that there are hydrogen bond interaction between bioactive compound in bpe and estrogen receptor (er)-α but less powerfull than estrogen and er-α interaction. in summary, bpe can act as an estrogen agonist, resulting in the enhancement of c-myc expression. keywords: banana peels extract (bpe), phytoestrogen, mammary gland, ovariectomized rats introduction estrogen is one of the important hormones in regulating of the menstrual cycle, reproduction, modulation of bone density and cholesterol transport (jordan, 2004). estrogen also stimulates proliferation of breast glandular epithelial cells by binding with estrogen receptors and inducing of estrogen receptors-mediated gene transcription (chen et al., 2000). therefore, large amounts of estrogen deficiency causes decreasing quality of women life. estrogen deficiency may affect breast’s health and beauty. it also triggers reproductive organs health disorder such as hot flashes (achadiat, 2003). estrogen deficiency usually can be treated by taking estrogen from outside of the body that is known as hormone replacement therapy (hrt). however, hrt is not only expensive but also causing breast and endometrial cancer. therefore, an alternative therapy that is relatively safer and cheaper is the use of phytoestrogen. phytoestrogen is a bioactive compound that has estrogen-like structure and activity (murkies et al., 1998). phytoestrogen binds to and activates estrogen receptors so that it gives estrogenic effects. sultana et al. (2008) proved that the bpe contains myricetin which is a flavonoid compound suspected as phytoestrogen. *corresponding author e-mail: meiyan_e@ugm.ac.id pratama, et al., 2011 indones. j. cancer chemoprevent., 2(1), 151-158 152 myricetin is able to activate erα and increase expression of c-myc so that it stimulates proliferation of breast epithelial cells mcf-7. bpe is suspected to affect breast glands development through the increasing c-myc level because it contains myricetin. estrogenic effects of phytoestrogen are influenced by affinity of phytoestrogen and estrogen receptor. the greater their affinity and interaction, the greater the expression of c-myc and cell proliferation. computational method using molecular docking approach performed as a method to determine the strength of interaction between myricetin and erα. this study was conducted to obtain scientific data about bpe estrogenic effects toward ovariectomized rats, that bpe could be used as a hormone replacement therapy agent. methods banana peels extract preparation ripe bananas were harvested from beran, sleman. banana’s peel was collected, dried using oven under temperature 40 o c, then it was made into powder using a blender (maspion) and extracted with 70% ethanol (maceration). macerate obtained was concentrated using a rotary evaporator to obtain viscous extract. animal study thirty six sprague dawley female rats 4050 days age with 86-118 grams of weight were obtained from unit pengembangan hewan percobaan (uphp) ugm. the rats were housed in plastic cages in a temperature and humiditycontrolled room (25-32 º c and 98% relative humidity) and were given free access to food and distilled water. after a 3-d adaptation period, 40 mice were either sham-operated (n =6) or ovx (n =24). treatment groups the rats were randomly divided into five groups of 6 each that were ovariectomized at the age of 50 days and another group wasn’t. ovariectomy (ovx) is an ovarian cutting process so that rats in the estrogen-deficient condition. one group of ovx mice received 0.5% cmc-na for two weeks as a solvent control, two group of ovx mice received 500mg/kgbw bpe and 1000 mg/kgbw daily for 2 weeks for two weeks, some ovx mice received 17-β-estradiol 2 μg/day per orally. at the end of the experiment, the mice were killed with petroleum eter. in each experiment, body weights were measured and mamary were isolated for the haematoxyllin and eosin (h&e) and immunohistochemistry analysis. haematoxyllin and eosin (h&e) staining the first right thoracic mammary gland from each animal was carefully dissected and fixed in neutral buffered formalin for routine histopathological analysis. briefly, mammary glands were dehydrated through a graded ethanol series, embedded in paraffin, and 5-_mthick sections cut and stained with hematoxylin and eosin. each slide was scored on a 4-point scale ranging from 0 (normal) to 4 (severe changes) by a pathologist blinded to treatment groups. histopathological changes were further scored according to distribution of changes (0.25 focal), 0.5 (locally diffuse), and 0.75 (diffuse). therefore, the most severely affected sections with severe diffuse pathological changes would be scored a maximum of 4.75. immunohistochemistry sections of 3 mm were obtained from each paraffin block of the organs. the section were washed and adhered for to poly-l-lysine-coated slides, incubated in prediluted blocking serum at 25 0 c, 10 minutes. the slides were stained for 1 hour at room temperature with primary ab c-myc (santa cruz). after washing with phosphate buffered saline (pbs), 100 µl of biotinylated universal secondary antibody (novocastra) were applied for 10 minutes at 25° c. the slide was incubated with streptavidin-biotin-complex for 10 minutes, 1 : 2 in pbs and 5% ab serum was added and washed with pbs. slides were incubated in 3,3 diaminobenzidin (dab) solution for 3-8 minutes and washed with aquadest. the slides were counterstained with hematoxyllin and eosin, 4 minutes. protein expression was assessed under light microscope. positive expression of c-myc will give a dark brown color in the nucleus area. molecular docking this method was done using licensed software named 2008.10 (chemical computing group inc.). the myricetin structure was built with molecule builder and ran energy minimalizing using am1 method. energy minimalizing step was run to get the most stable molecule by finding the conformation with lowest energy. erα 3-d structure was taken from protein data bank (www.rcsb.org). the docking molecular was run pratama, et al issn : 2088-0197123 153 between receptor active site and ligand (myricetin). the output of this method is to get ligand-receptor interaction with lowest score which has highest stability and identified ligandreceptor interaction type. data analysis. cell morphology staining with (h&e) and c-myc expression were analyzed by excell ms office 2003 and semi-log analysis (spss 11.5). one way anova was used to assess concentration (p<0.05) then post-hoc comparissons were made using tuckey’s significant different test. molecular docking was done to know myricetin-er α interaction. myricetin-er α interaction is getting stonger when the score is getting down. results effect of banana peels extract in ovariectomized mice mamary gland quantitative he analysis, carried out in a wide series of rat mamary glands showed that ovariectomized made any differences on mice mamary gland lobulus (fig.1b). mamary gland growth was greater in mice treated bpe 1000 mg/kgbw (fig.1f) and treated estradiol (fig.1d) when compared with that of ovariectomized control. bpe-treated (fig.1e) and cmc-na treated (fig.1c) animals were not significantly different from each other. however, size of lobulus cell on mice treated bpe 1000 mg/kgbw and estradiol are relatively bigger than lobulus on nonovariectomized mice. statistically (p>0.05), ovariectomized on ovariectomy mice decreased the amount of lobulus even it was not significant toward nonovariectomy mice groups (fig.2). estradiol significantly increased the amount of cell lobulus. on bpe 500 mg/kgbw treated groups, increased lobulus cell unsignificant towards cmc-na treated groups. bpe 1000 mg/kgbw enhanced lobulus cell proliferation significantly towards ovariectomized control (fig.2). no significant differences between the amount of lobulus cell among mice treated bpe 500 mb/kgbw, bpe 1000 mg/kgbw and estradiol itself. immunohistochemistry expression of c-myc on mamary gland was determined by immunohistochemstry assay. the cell which express c-myc will be brown nearby nucleus area. the higher the colour intensity, the higher the c-myc expression. the expression of cmyc was positively on novx groups (fig.3a), estradiol groups (fig.3d) and bpe 1000 mg/kgbw (fig.3f) due to brown spot with high intensity. cmyc appearance on mamary gland indicated cell proliferation. quantification of c-myc expression on mamary lobulus cell (fig.4) showed that lobulus cell in ovx groups expressed c-myc lower than novx groups hile estradiol group expressed the highest c-myc. statistically, c-myc expression on novx and estradiol were unsignificant. over all, anova and tuckey test showed unsignificant enhancement of c-myc expression on each groups. probably, the mechanism of mamary gland proliferation on bpe treated rats took another pathway. indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 154 figure 1. mice mamary gland hystology (h&e stainning). the experiments was done under light microscope with 100x magnification. non-ovariectomized groups (a), ovariectomized groups (b), cmc-na groups (c), estradiol groups (d), bpe 500 mg/kgbb groups (e) and bpe 1000 mg/kgbw groups (f). sign shows lobulus cell. figure 2. quantitative analysis of mamary gland lobulus cell (h&e). statistical analysis was done using anova dan uji tuckey (p<0,05). amount of lobulus was shown from average ± standard of error (se) of 3 experiment sign (*) showed significant differences. indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 155 figure 3. determined c-myc expression by immunohistokimia assay. the experiments was done under microscope with 100x magnification. non-ovariectomized groups (a), ovariectomized groups(b), cmc-na groups (c), estradiol groups (d), bpe 500 mg/kgbb groups (e) and bpe 1000 mg/kgbw groups (f). brown spot in cytoplasm and perinuclear show c-myc expression. sign show lobulus cell which expressed c-myc. figure 4. quantitative analysis of c-myc expression on mamary gland lobulus cell (immunohistochemistry). statistical analysis was done using anova dan uji tuckey (p<0,05). amount of c-myc expression on lobulus was shown from average ± standard of error (se) of 3 experiment indonesian journal of cancer chemoprevention, february 2011 issn: 2088–0197 e-issn: 2355-8989 156 molecular docking the interaction between er α and myricetin (ligand) was predicted using molecular docking software. er α structure was reached from protein data bank with code 1a52. the method of docking validation use estrogen receptor and native ligand as subjects. native ligand is estrogen (fig.5) with root mean square deviation (rmsd) = 0,329372. rmsd score < 2 show that the method run with high validity. this method could predicted the position of ligand on receptor active site, ligand-receptor binding and the strength of ligand-receptor binding. the ligand on this method is myricetin (fig.6) which is the bioactive compound on bpe. the active sited receptor placed by myricetin and estrogen (fig.7) showed that both compound placed the same site on receptor but with different posed. . figure 5. structure of estrogen figure 6. structure of myricetin the interaction between estrogen and erα (fig.7a and 8a) is two interaction of hydrogen bonding. the hydrogen bond occured between oxygen on 17-hydroxy groups with hydrogen on hystidin 524 residu (fig.7a) and between hydrogen on 3-hidroxy groups with oxygen carbonil on 353 glutamic acid residu (fig.7a). interaction between myricetin and erα (fig.7b and 8b) is two interaction of hydrogen bonding. the hydrogen bond occured between hydrogen on 4-hidroxy groups with oksignen carbonil on histidin 353 residu (fig.7b) and between hydrogen on 5-hydroxy groups with oxygen carbonyl on 346 leucin residu (fig. 7b). . (a) (b) figure 7. 3d docking result appearances. estrogen (a) and myricetin (b) bind in the same place on estrogen receptor alfa with different pose. pratama, et al issn : 2088-0197123 157 (a) (b) figure 8. 2d profile of ligand-amino acid residu on receptor. hydrogen bond interactions between hystidin 524 and glutamic acid 353 group of estrogen (a) whereas myricetin (b) make a hydrogen bond interaction between glutamic acid 353 and leusin 346. sign showed hydrogen bond between ligand and amino acid residu on receptor. the strength of ligand-receptor bonding was shown by docking score using london dg scoring so that ligand bond with certain poses could be estimated. the lower the score, the more optimal ligand pose so that the bonding was getting stronger and stable. the molecular docking result showed that estrogenerα interaction gives lower score than myricetinerα interaction (table 1). it showed that myricetinerα interaction is less powerfull than estrogenerα interaction. table 1. molecular docking score of estrogen receptor (erα). ligand score (kkal/mol) estrogen -25,1793 myricetin -15,5834 discussion estrogenic effect as in vivo could be examined from the amount of mamary lobulus cell and c-myc expression upregulation. estradiol enhance the amount of mamary lobullus cell significantly. bpe 500 mg/kgbw treated groups, mammary lobulus cell increased unsignificantly towards cmc-na treated groups. bpe 1000 mg/kgbw enhanced lobulus cell proliferation significantly towards ovariectomized control. expresssion of c-myc was high on novx, estradiol, and bpe 1000 mg/kgbw groups. in silico experiments with molecular docking showed model interaction between estrogen receptor with the ligand. the result showed that myricetin could have such an interaction with erα through hydrogen bonding and placed the same active site as estrogen. however, myricetin-erα bonding was less powerfull than estrogen-erα. this phenomena showed that estradiol group has higher lobulus cell amount and c-myc expression than bpe 1000 mg/kgbb. however, the interaction of agonists and antagonists could not be ascertained with this computational method and there is the possibility of other flavonoids which are more capable to produce estrogenic effect, or perhaps other flavonoids blocked myricetin affinity to erα. thus, this needs to be proven by further studies with other compounds contained in bpe test and molecular docking of all flavonoid compounds in bpe. probably, bioactive compound on bpe (myricetin) is a phytoestrogen due to interaction with estrogen receptor so that induces mamary lobulus cell proliferation. according to sultana et al. (2008), one of bpe bioactive compound is myricetin. maggiolini et al. (2005) on their research said that myricetin could enhance pratama, et al., 2011 indones. j. cancer chemoprevent., 2(1), 151-158 158 mamary lobulus cell proliferation. probably, myricetin promotes mamary epithelial growth via certain pathway and activates estrogen receptor by binding with the receptor. those activated receptor are going to bind with estrogen response element (ere) (van der woude et al., 2005) and induces estrogen responsive gene expression, one of them is c-myc expression. cmyc and cyclin d are g1 phase regulatory protein so that those could induces phase g1 cycle cell (gardner et al., 2003). some protein such as c-myc and cyclin d play a role in transition step between g1 phase and s phase. those induces cyclin e-cdk2-p130 complex formation and cyclin e-cdk2-p21 inactive complex deformation (doisneau-sixou et al., 2003). cyclin e-cdk2 complex that release from p21, will bind with p130 that phosporilated e2f-prb inactive complex become free e2f stimulating s phase. but, the proposed mechanism needs to be explored further to know the exact mechanism of bpe. overall, this research showed that bpe has an estrogenic effect proved by enhancing mamary lobulus cell and c-myc expression. so that, bpe is a potential herbs to be explored further as estrogen-deficiency drugs. probably myricetin promotes mamary epithelial growth via certain pathway that needs to be explored further. conclusion bpe dose 1000 mg/kgbw enhances mamary gland development and induces c-myc expression significantly. the molecular docking showed the bioactive compound on bpe have a good interaction with erα through hydrogen bound and placed the same active site with estrogen but estrogenerα binding is much stronger and stable. acknowledgement this work was supported by due like/ppkb ugm (peningkatan pertumbuhan kepemimpinan berkualitas) 2009. references achadiat, c.m., 2003, fitoestrogen untuk wanita menopause, http://situs.kesrepro.info/aging/jul/2003/ag 01.htm, accessed september 2010. chen, x., danes, c., lowe, m., thaddeus, w., herliezek and keyomarsi, k., 2000, activation of the estrogen-signaling pathway by p21wafi/cipi in estrogen receptor-negative breast cancer cells, j. natl. cancer inst., 92, 17,1403-1413. doisneau-sixou, s.f., 2003 estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells. endrocrine-related cancer, 10, 179-186. gardner, l., lee, l. and dang, c., 2002, the cmyc oncogenic transcription factor. encyclopedia of cancer, 2. jordan, c.v., 2004, selective estrogen receptor modulation: concept and consequences in cancer, 5, 207-213. maggiolini, m., recchia, b., catalano, v., carpino, r.v., rossi, r. and ando, s., 2005, the red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor α in human breast cancer cells, journal of molecular endocrinology, 35, 269281. murkies, a.l., gisela, w. and susan, r.d., 1998, phytoestrogen, journal of endrocrinology and metabolism, 83(2), 297-303. sultana, b., farooq, a., muhammad, r.a. and shahzad, a.s.c., 2008, antioxidant potential of extracts from different agro wastes: stabilization of corn oil, grasas y aceites, 59(3), 205-217. van der woude, h., ter veld, m.g., jacobs, n., van der saag, p.t., murk, a.j. and rietjens, i.m., 2005, the stimulation of cell proliferation by quercetin is mediated by the estrogen receptor, mol. nutr. food res., 49(8), 763-771. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22van+der+woude+h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22ter+veld+mg%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22jacobs+n%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22van+der+saag+pt%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22murk+aj%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22rietjens+im%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22rietjens+im%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'mol%20nutr%20food%20res.'); javascript:al_get(this,%20'jour',%20'mol%20nutr%20food%20res.'); secang’s heartwood (caesalpinia sappan l indonesian journal of cancer chemoprevention, june 20016 issn: 2088–0197 e-issn: 2355-8989 60 heartwood of secang (caesalpinia sappan l.) ethanolic extract show selective cytotoxic activities on t47d and widr cells but not on hela cells erlina rivanti, bani adlina, ika nurzijah, cyndwika ayu, adam hermawan * cancer chemoprevention research center, faculty of pharmacy, universitas gadjah mada abstract the present study investigate the selectivity of heartwood of secang ethanolic extract (see) on t47d breast cancer cells, widr colon cancer cells, and hela cervical cancer cells, compared to vero normal epithelial cells. the cytotoxic effect was evaluated by using mtt assay with 24-hour treatment to get ic50 values. selectivity was evaluated by using selectivity index (si). see had a potent cytotoxic activity on t47d and widr cancer cells (ic50<100 µg/ml). ic50 value of hela cancer cells was observed on moderate cytotoxic (100<ic50<1000 µg/ml). see demonstrated more selective to t47d and widr than vero cells (si>3), while in hela cells is not selective (si<3). this result indicating its potential of caesalpinia sappan as a chemopreventive agent in cancer therapy. keywords: cancer, selectivity, secang, t47d, widr, hela, vero introduction currently, a chemopreventive agent based on natural materials has been developed. it is triggered by the fact that the treatment of cancer using chemotherapeutic agents tends to cause resistance of cancer cell, resulting the majority of cancer treatment failure (nobili, et al., 2009). in addition, in clinical application, some classical chemotherapeutics such as doxorubicin and 5-fluorouracil showed side effects such as cardiotoxicity and myelosuppression (bowles, et al., 2012, santos, et al., 2010). this is due to the lack of selectivity of the chemotherapeutics against cancer cells, thus causing disruption of homeostasis in the body. one of the potential natural products as a chemopreventive agent is secang (caesalpinia sappan l.). heartwood of secang and its active compound, brazilin and brazilein, proved has cytotoxic effect and induce apoptosis on cancer cells. study on antioxidant compound from c. sappan succeed to isolate 1',4'-dihydrospiro[benzofuran-3(2h),3'-[3h-2]benzopyran]1',6',6',7'-tetrol and 3-[[4,5-dihydroxy2(hydroxymethyl)phenyl]-methyl]-2,3-dihydro3,6-benzofurandiol (safitri, et al., 2003). park, et al. (2002) and wicaksono, et al. (2008) reported that secang has cytotoxic effects on hep g2 and hep 3b cancer cells. extract of c.sappan heartwood performed anticancer activity on hnscc4 and hnscc31 head and neck cancer cells (kim, et al., 2005). ethanolic extract of c. sappan also showed potent radical scavenging activity of reactive oxygen species (ros) and reactive nitrogen species (rns) (lee, et al., 2010). methanolic extract of c. sappan exhibited cytotoxicity (ic50<20 μg/ml) against hepg2 liver, mcf-7 and mda-mb-231 breast, and a549 lung cancer cell lines (lai, et al., 2011). yen, et al. (2010) reported that brazilein isolated from secang showed potent cytotoxic effects on hep g2 and hep 3b liver cancer cells, mda-mb-231 and mcf-7 breast cancer cells, and a549 lung cancer cells. based on those data the potency of secang as chemopreventive agent need to be explored in different cell lines. this present study was performed with the aim of determining of cytotoxicity of heartwood of secang ethanolic extract (see) on t47d breast cancer cells, widr colon cancer cells, and hela cervical cancer cells and its selectivity compared to vero normal cells. *corresponding author e-mail: adam_apt@ugm.ac.id rivanti, et al., 2017 indones. j. cancer chemoprevent., 7(2), 60-67 61 the scientific data regarding the activity of see as a selective agent could be the basis of the use of natural products as a chemopreventive agent in cancer therapy. materials and methods plants material heartwoods of secang were collected from wonosari, yogyakarta, indonesia. plants were identified at laboratory of plants taxonomy, faculty of biology, universitas gadjah mada, yogyakarta. the extract was prepared by maceration methods using 70% ethanol, then the filtrate was concentrated by vacuum rotary evaporator. cell culture in the study, t47d cells, widr cells and hela cells were obtained from prof. masashi kawaichi (nara institute of science and technology, naist, japan), while vero cells were obtained from cancer chemoprevention research center, faculty of pharmacy, ugm. cells were cultured at 37 o c in a humidified incubator, 5% co2, in suitable medium for each cells (dmem (gibco) for t47d and hela cells, rpmi 1940 (gibco) for widr cells, and m199 (gibco) for vero cells) supplemented with 10% fetal bovine serum (fbs) (gibco), 10,000 units/ml penicillin-10,000 µg/ml streptomycin (gibco). cytotoxicity assay cytotoxicity of see on t47d, widr, hela, and vero cells were determined using mtt assay (mosmann, 1983). cells were distributed into 96-well plate with the density of 1x10 4 cells/well, then incubated in 37˚c incubator supplemented with 5% co2 for 24 hours. see was diluted in culture medium using dimethyl sulfoxide (dmso) as co-solvent with concentration not more than 0.5 % v/v. after 24 hours incubation, the culture medium was removed, followed by washing with phosphate buffered saline (pbs). then, 0.5 mg/ml of mtt (3-[4,5-dimethyl thiazole-2-yl(2,5diphenyltetrazoliumbromide)]) in pbs was added, followed by 4 hours incubation in 37˚c with 5% co2. after that 10% v /v sds in hcl 0.1n as stopper reagent was then added. the plate was then kept with protection from light overnight, continued with absorbance determination (λ 595 nm) using elisa reader (bio-rad). data analysis the cell viability was calculated based on the formula as follow: % of viable cells = [abs. of treated cells – abs. of control medium] x100%. [abs. of control cells – abs. of control medium] the selectivity of see on each cancer cell lines was evaluated using si (selectivity index) value based on formula as follow (prayong, et al., 2008): ic50 on vero cells si = ic50 on cancer cells see is selective if si value>3, but not selective if si<3. statistical analysis all data are expressed as mean and standard deviation (sd) (n=3). statistically significant difference was determined by analysis of variance (anova) followed by post-hoc comparisons using tuckey’s significant difference test. statistical significance was considered at p<0.05 (spss 17.0). results the cytotoxic assay was performed to determine the inhibition of see on the growth of t47d breast cancer cells, widr colon cancer cells, hela cervical cancer cells, and vero normal epithelial cells. cell viability was examined using mtt assay after 24 hours incubation. treatment of see decreased cell viability of t47d, widr, hela, and vero in dose-dependent manner (fig. 1). cells morphology was also observed (fig. 2), comparing the control cells to treated cells. treatment of see altered the morphology of t47d, widr, hela and vero cells. rivanti, et al., 2017 indones. j. cancer chemoprevent., 7(2), 60-67 62 figure 1. effects of see on cell growth. (a) t47d cancer cells; (b) widr cancer cells; (c) hela cancer cells; (d) vero normal cells. see showed dose-dependent effect on cells growth. the assay was performed by incubating 5x103 cells/well with a serial concentration of see. after 24 hours, mtt reagent was added to evaluate cell viability as described in methods. cell viability profile is shown as average±standard of deviation (sd) of 3 experiment. 0 10 20 30 40 50 60 70 80 90 100 10 25 50 100 150 200 concentration of see (µg/ml) c e ll v ia b il it y ( % ) t47d 0 20 40 60 80 100 120 140 160 1 2,5 5 10 15 20 30 concentration of see (µg/ml) c e ll v ia b il it y ( % ) widr 0 20 40 60 80 100 120 10 25 50 100 200 300 concentration of see (µg/ml) c e ll v ia b il it y ( % ) vero a b c d 0 20 40 60 80 100 120 140 50 100 200 300 400 500 concentration of see (µg/ml) c e ll v ia b il it y ( % ) hela rivanti, et al., 2017 indones. j. cancer chemoprevent., 7(2), 60-67 63 a b c d figure 2. morphological changing caused by see treatment comparing to control cell. (a) t47d cancer cells (b)widr cancer cells; (c) hela cancer cells; (d) vero normal cells. morphological changing representing cells’ dead show by red arrow. cell morphology was examined by using inverted microscope with magnification 400x control sel 10 µl 50 µl 150 µl control 1 µl 15 µl 30 µl control sel 25 µl 100 µl 400 µl control sel 10 µl 100 µl 200 µl rivanti, et al., 2017 indones. j. cancer chemoprevent., 7(2), 60-67 64 table 1. ic50 and si value cells ic50 (µg/ml) si t47d 36 3.72 widr 30 4.47 hela 327 0.41 vero 134 see showed moderate to high cytotoxicity on t47d, widr, and hela cells with ic50 value 36 µg/ml, 30 µg/ml, and 327 µg/ml, respectively (table 1). the highest potency was showed on widr cells. while the lowest ic50 value of see was 134 µg/ml on vero cells, as we expected. in order to evaluate the selectivity of see, we calculated the selectivity index (si) value of see on cancer cells. si value was calculated using a formula based on previous study (prayong, et al., 2008), by comparing the ic50 value of see on vero normal cells to ic50 value on cancer cells (table 1). see showed selective cytotoxicity on t47d and widr with si value more than 3. on the other hand, see did not selective on hela cells with si less than 3. discussion in this study, we observe the cytotoxic properties of secang’s heartwood ethanolic extract (see) and its selectivity directed t47d breast cancer cells, widr colon cancer cells and hela cervical cancer cells. development of molecularly targeted agent that more specific kills cancer cells and have less damage to normal cells is still the main goal in anticancer drug discovery (nurulita, et al., 2011). many researchers are now interested in examining the use of herbal medicines as a health care method. see showed moderate to high potency as chemopreventive agent based on its cytotoxic effect on t47d, widr, and hela cells. since see had high potency of cytotoxicity, it is important to reveal the selectivity of the extract. see showed moderate cytotoxicity on vero cells. to determine the see selectivity, si was calculated. see showed selective cytotoxic activity on t47d and widr cells (si>3), but not selective on hela cells (si<3). the differences activity of see on several cells might caused by differences in cells characteristic or difference target of action. t47d cells have a mutation of p53 and express estrogen receptor (er) (schafer, et al., 2000). compounds isolated from methanolic extract of c. sappan exhibited partial antiestrogenic activity on mcf-7 cells (lai, et al., 2011). upregulation of p21 might occurred through p53 independent pathway leading to inhibition of cell cycle (kim, et al., 2012). extract of c. sappan also showed anticancer activity on hnscc4 and hnscc31 cells (head and neck cancer cells) upregulation of p21waf1/cip1 (kim, et al., 2005). see proposed to inhibit t47d cell viability through antiestrogenic activity, and inhibition cell cycle, but the mechanism need to be explored further. widr cells show overexpression of cox-2 and mutant of p53 (palozza, et al., 2005; bijman, et al., 2008). apoptosis occured through p53 independent pathways, including activation of p73 (levrero, et al., 2000). ethanolic extract of c. sappan reduced levels of inflammatory cytokine and expression of cox2 and transcription factor of nf-κb p65 in paw cartilage of cia rats (wang, et al., 2011). brazilin inhibits cell cycle through activation p21 and p27 on u266 cells (kim, et al., 2012). sappanchalcone, a flavonoid isolated from c. sappan, induces apoptosis through inhibition of p38, erk, jnk, and nf-κb signaling on oral cancer cells (lee, et al., 2011). see proposed to reduce widr cell viability through downregulation of cox-2 and nf-κb, but the mechanism need to be clarified more details. hela cells have hpv 18 sequences that express e6 and e7 causing p53 degradation (goodwin and dimaio, 2000). zheng, et al. (2006) reported that the induction of apoptosis on hela cells is mediated by upregulation bax and downregulation of bcl-2. see showed moderate cytotoxicity on hela cells. compounds of see might be selectively effective on receptor on t47d and widr cells rather than receptor on hela cells. binding of fasl in fas receptor is responsible for p53 independent apoptosis (reuter, et al., 2008). hela cells induce fasl expression via corticotropin-releasing hormone (crh) (taliouri, et al., 2012). higher-affinity antibodies (fasl) performed a significant rivanti, et al., 2017 indones. j. cancer chemoprevent., 7(2), 60-67 65 decrease, rather than an increase, in agonist activity at the fas receptor (chodorge, et al., 2012). compounds in see might be influence affinity of fasl and fas receptor, but the mechanism must be explored in the next study. extract of heartwood of secang also safe to be consumed. secang has been used as ingredients of traditional beverage in central java, indonesia. the extract of c. sappan wood showed neither toxicity in terms of general behavior change, mortality, change in gross appearance of internal organs nor produce any acute and subacute toxicity in both female and male rats (sireeratawong, et al., 2010). in vivo study of ethanol extract of c.sappan l on mice fed a high-cholesterol diet showed increase of hepatic antioxidative indicators, as well as decrease of intracellular ros, lox-1, and nuclear translocation of nfκb on in vitro study in cultured human umbilical vein endothelial cells (huvecs) (lee et al., 2010). the results of this present study, adds the scientific evidence of c. sappan considering its potency as a cancer chemopreventive agent. conclusion heartwood of secang ethanolic extract (see) possesses selectivity effect on t47d and widr cancer cells but not on hela cancer cells. further caesalpinia sappan reveals prospective properties to be developed as cancer chemopreventive agent. acknowledgement the authors thank dp2m dikti for 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