Bangladesh Journal of Pharmacology Research Article Acetoxyroyleanone exhibits selec- tive anti-cancer effects and induces apoptosis in human colon carcino- ma cells through the mediation of NF-κB and caspase-3 signalling pathways BJP Introduction Colorectal carcinoma is a cancer from uncontrolled cell growth in the colon or rectum or in the appendix. The number of colorectal carcinoma cases increased to 1.3 million with more than 7 lakh deaths globally as per latest global cancer statistics (Jemal et al., 2011). Factors like cell proliferation, cancer cell migration, angiogenesis and inflammation play key roles in colorectal carcinoma (Rupnarain et al., 2004; Kinzler and Vogelstein, 1996; Rodrigues et al., 1990). Conventional chemotherapy invol- ving 5-fluorouracil, leucovorin, capecitabine and irinote- can regimens for the treatment of colorectal cancer have inadequate efficacy and are accompanied with significant severe adverse effects. Acetoxyroyleanone is an abietane diterpenic natural product usually isolated from various plant species particularly from Salvia species such as Salvia nemorosa and S. martiusii (da Araújo et al., 2006; Vlasova et al., 1969). Acetoxyroyleanone contains a quinone moiety as its key structural feature. It has been reported that abietane diterpenes are often reported to exhibit cyto- toxic effects on cancer cell lines. It was reported that the cytotoxic activity of acetoxyroyleanone was related to the inhibition of DNA synthesis and induction of apop- A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2016; 11: 141-147 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract The objective of the current study was to evaluate the antiproliferative and apoptotic activities of acetoxyroyleanone against various cancer cells along with studying its effect on chromatin condensation, NF-κB and caspase-3 expressions and cell migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell viability while as fluorescence microscopy revealed the effects of acetoxyroyleanone on cellular morphology of Colo-205 cells. Western blotting revealed effects on NF-κB and caspase-3 expressions. The results revealed that acetoxyroyleanone induced potent and dose-dependent antiproliferative effects against a range of cancer cell lines with Colo-205 being the most susceptible cell line. However, it required six to eight times higher concentration of acetoxyroyleanone to induce 50% cell death in normal epithelial (fR-2) cell line. Further, following acetoxyroyleanone treatment to Colo-205, it was observed that acetoxy- royleanone induced substantial down-regulation of NF-κB and up-regulation of caspase-3 expressions. In addition, acetoxyroyleanone impairs cell migra- tion, chromatin condensation, cell shrinkage and membrane blebbing. Article Info Received: 27 July 2015 Accepted: 26 August 2015 Available Online: 1 January 2016 DOI: 10.3329/bjp.v11i1.24339 Cite this article: Zhong XJ, Jia H, Hu JL, Le F, Zhong XM. Acetoxyroyleanone exhibits se- lective anti-cancer effects and induces apoptosis in human colon carcinoma cells through the mediation of NF-κB and caspase-3 signalling pathways. Bangladesh J Pharmacol. 2016; 11: 141 -47. Acetoxyroyleanone exhibits selective anti-cancer effects and induces apoptosis in human colon carcinoma cells through the mediation of NF-κB and caspase-3 signalling pathways Xiao-Jun Zhong1, Hui Jia2, Ji-Long Hu3, Fei Le4 and Xiao-Ming Zhong5 1Department of Oncology, The First Affiliated Hospital of Nanchang University, Nanchang 330 006, Jiangxi Province, China; 2Departments of Internal Medicine-Oncology，Shandong Cancer Hospital and Institute, Jinan 250 117, Shandong Province, China; 3Department of Tumor Abdominal Surgery, Jiangxi Tumor Hospital, Nanchang 330 029, Jiangxi Province, China; 4Department of Head and Neck Surgery, Jiangxi Province Tumor Hospital, Nanchang 330 029, Jiangxi Province, China; 5Department of Tumor Radiotherapy and Chemotherapy, Jiangxi Tumor Hospital, Nanchang 330 029, Jiangxi Province, China. This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and per- form the work. You must attribute the work in the manner specified by the author or licensor tosis (da Araújo et al., 2006). Acetoxyroyleanone has also been reported to induce cytotoxic effects in pancreatic cancer cell line (Fronza et al., 2012). Despite this, till now, very little information exists in the literature regarding the cytotoxic molecular mechanism of action of acetoxyroyleanone. In the present study, the molecular mechanism of action of acetoxyroyleanone is reported by studying its effects on apoptosis induction, NF-kB and caspase-3 signalling pathways. Materials and Methods Chemicals and source of antibodies and kits Growth medium (MEM/RPMI), fetal calf serum, trypsin, penicillin, streptomycin, dimethyl sulfoxide, RNase, proteinase K, RIPA Buffer, bisacrylamide, SDS, ([3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]) dye, acrylamide, ammonium persulfate, 2-mercapto- ethanol, and Tris-base were obtained from Hangzhou Sijiqing Biological Products Co. Ltd, China. Chemilumi- nescent Western blotting kit (Millipore), Quanti Pro BCA assay kit, 96 and 6 well plate (Iwaki), triton X (Hi-Media), Tris-EDTA (Hi-Media), acetic acid (Rankem), ELISA plate reader (Bio-Rad). NFκB (p65), caspase-3 antibodies were purchased from Millipore Pvt. Ltd. Acetoxyroy- leanone was purchased from Sigma Chemical Company (USA), and 100 mg/mL solution dissolved in dimethyl sulfoxide was stored at -20ºC prior to use. Cell lines, growth medium and treatment conditions Human cancer cell lines; prostate (PC-3), leukemia (THP-1), colon (Colo-205, Caco-2), breast (T47D,MCF- 7), pancreatic (MiaPaca-2) and normal epithelial (fR-2) were procured from Shanghai Institute of Cell Resource Center of Life Science (China). Cells were grown in Minimum Essential Medium and Roswell Park Memo- rial Institute medium supplemented with 10% fetal calf serum and 1% penicillin. Penicillin was dissolved in phosphate buffered saline and sterilized by filtering through 0.2 μm filter in laminar air flow hood. Cells were cultured in CO2 incubator (New Brunswick, Galaxy 170R, eppendroff) with an internal atmosphere of 95% air and 5% CO2 gas and the cell lines were maintained at 37ºC. The media was stored at low temperature (2-8ºC) and the medium for cryopreser- vation contained 20% fetal calf serum and 10% dime- thyl sulfoxide in the growth medium. MTT assay for cell viability evaluation Cell viability was measured using [3-(4, 5-dimethyl- thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT) assay. Different cell lines were seeded in 200 μL of Roswell Park Memorial Institute medium-1640 medium into 96-well plates, and cultured overnight. Then the medium was replaced with fresh RPMI-1640 or the same media containing different concentrations of acetoxyroyleanone. After a further incubation for 24 hours, 30 μL of MTT (2 mg/mL) was added to each well followed by 3 hours incubation. The medium was discarded and 170 μL of dimethyl sulfoxide was added to each well, and incubated for 30 min. The OD490 nm was measured. The cell viability index was calculated according to the formula: (Experimental OD value/control OD value) × 100% Cytotoxicity was expressed as the concentration of acetoxyroyleanone inhibiting cell growth by 50% (IC50 value). Preparation of whole cell lysates and Western blot analysis Colo-205 (1 x 105 cells/mL/well) cells were treated with acetoxyroyleanone at 0, 5, 10, 20 and 30 µM were suspended in cold RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1%SDS, 50 mM Tris, PH 8.0) for 30 min on ice. The lysates were vortexed and centrifuged at 12,000 xg for 10 min. Supernatant thus obtained was whole cell lysate and was stored at -20ºC for further use. Protein content was measured using bovine serum albumin as standard. 1 mg/mL protein standard was taken and samples with unknown concentrations were plotted in a linear range of 0.5 to 30 μg/mL of the protein concentration and absorbance measured at 562 nm. The above protein lysates were subjected to discontinuous SDS-PAGE at 100 V and electro transferred topolyvinylidene difluoride membrane (Millipore) for 2.5 hours at 120 V at 4ºC. The membrane was blocked with 2% skimmed milk in phos- phate buffered serum for 1 hour. After blocking, the membrane was probed with specific primary antibody for overnight at 4ºC followed by 2 times washing with tris-buffered saline for 5 min each. A dilution of secon- dary antibody (mouse and rabbit) conjugate was added for 1 hour of incubation and signals were detected using Millipore chemiluminescent Western blotting kit and analyzed using X-ray film. Cell migration assay Cell monolayer (90% confluent) was allowed to become quiescent in medium with 0.1% dialyzed fetal bovine serum for 24 hours. Then cells were scraped to make a straight line wound and treated with acetoxyroyl- eanone for 48 hours. Photographs were taken at 48 hours and lengths of the wound were determined by Image J (version 1.46) software. Statistical evaluation The results of three independent experiments were expressed as the mean ± SD. Statistical evaluation was performed using an un-paired t-test. 142 Bangladesh J Pharmacol 2016; 11: 141-147 Results In vitro cytotoxic effect of acetoxyroyleanone against different human cancer cell lines In this study, in vitro inhibitory effect of acetoxyroyleanone was evaluated using the MTT viability assay against a range of cancer cell lines and IC50 after 48 hours was calculated. Initially, acetoxyroyleanone was screened (Table I) against leukemia (THP-1), prostate (PC-3), breast (MCF-7,T47D), pancreas (MiaPaca-2), colon (COLO-205, Caco-2) cancer cell lines including normal epithelial cell (fR-2) at indicated concentration (5, 10, 20, 30 and 50 µM) for 48 hours. Acetoxy- royleanone (Figure 1) shows concentration-dependent inhibi- tory effect on cell proliferation against THP-1, T47D, MiaPaca- 2 MCF-7 and Colo-205 cancer cell lines and most potent inhibition against Colo-205. The cell viability assay revealed that in Colo-205, acetoxyroyleanone (5–50 µM) increased growth inhibition of 96, 82 and 79% at a concentration of 50, 30 and 20 µM (Figure 2), with the calculated IC50 value of 12 μM and 10 nM for BEZ-235 which were used as positive control (Table I). However, it required six to eight times higher concentration of acetoxyroyleanone to induce 50% cell death in normal epithelial (fR-2) cell line. Interestingly, these results also depicted that acetoxyroyleanone showed more efficiency against Colo-205 as revealed by their relative IC50 values. Effect of acetoxyroyleanone treatment on NF-κB and caspase-3 expression in Colo-205 cell line In next experiment, the effect of acetoxyroyleanone on NF-κB and caspase-3 protein expression levels by Western blotting was observed. Following acetoxy- royleanone (10 and 30 µM) treatment for 48 hours, Western blot analysis revealed that acetoxyroyleanone decreased expression level of NF-κB (p65) and increa- sed caspase-3 expression level (Figure 3). However, significant effect on both proteins was observed at 30 µM acetoxyroyleanone concentration as compared to untreated and BEZ-235 (10 nM) (positive control) (Figure 4), suggesting NF-κB down-regulation and caspase-3 up-regulation by acetoxyroyleanone. Treatment of acetoxyroyleanone inhibits cell migration of Colo-205 cell monolayers Wound closure experiments were performed to estab- lish the inhibitory effect of acetoxyroyleanone in Colo- 205 cell migration. As shown in Figure 5, the wound got almost healed in untreated and slightly so at the lower concentrations (10 µM) of acetoxyroyleanone. However, at higher concentration (30 µM) invasiveness of Colo- 205 cells showed potent cell migration inhibition. Acetoxyroyleanone treatment results in significant inhibition of Colo-205 cells invasion at a concentration of 30 µM compared to negative control (0 µM) and positive control (BEZ-235, 10 nM) (Figure 6). Discussion In the present study, an interesting correlation was discovered for the first time between various regulatory and phenotypic events of acetoxyroyleanone with apoptosis. First, the growth inhibitory and cytotoxicity effect of acetoxyroyleanone against a panel of human cancer cell lines which include leukemia (THP-1), prostate (PC-3), breast (MCF-7, T47D), pancreas (MIAPaca2), colon (Colo-205, Caco-2) including normal Table I IC50 value of acetoxyroyleanone against different cancer cell lines including normal epithelial cell line Tissue Cell Line IC50 (µM) (Acetoxyroyleanone) IC50 (nM) (BEZ-235) Leukemia THP-1 27 - Prostate PC-3 >50 - Breast T47D 24 10 Breast MCF-7 28 - Pancreas MiaPaCa-2 25 10 Colon Colo-205 12 10 Colon Caco-2 >50 - Normal fR-2 >50 - Figure 1: Chemical structure of acetoxyroyleanone OAc O O OH Bangladesh J Pharmacol 2016; 11: 141-147 143 epithelial cell (fR-2) was evaluated. Particularly, it was found that acetoxyroyleanone inhibits cell growth and more significantly showed concentration-dependent inhibition against a panel of cancer cell lines tested. Interestingly, maximum and potent growth inhibition following acetoxyroyleanone were observed at 50, 30 and 20 µM in human colon cancer cell line i.e. Colo-205. Keeping this in view, the IC50 value of acetoxyroyl- eanone against all shown human cancer cell lines by cell viability assay was evaluated. The calculated IC50 values of acetoxyroyleanone were of the order of 27, 24, 28, 25 and 12 µM for 48 hours incubation in the case of THP-1, MCF-7, T47D, MiaPaca-2 and Colo-205 respectively. While as no significant cytotoxic effects were found in the normal epithelial cell line (fR-2). Overall, these results depicted that acetoxyroyleanone showed signifi- cant effect against Colo-205 colon cancer cell prolifer- ation as reflected by relative IC50 value. Since the NF-κB pathway is important for cell survival, proliferation, cell cycle progression and migration. Acti- vation of NF-κB pathway affects regulation of prolifera- tive, anti-apoptotic, pro-apoptotic and cell cycle regula- tory molecules and therefore results in cell prolifera- Figure 2: Growth inhibitory effect of acetoxyroyleanone against panel of cancer cell lines including normal epithelial cell line. Cells grown in 96 well plate was treated with different concentration of acetoxyroyleanone (5, 10, 20, 30 and 50 µM). Data repre- sent mean ± SD (n=3) of three repeats Figure 3: Western blot analysis reveals decrease in NF-κB (p65) and increase in caspase-3 expression at 10 and 30 µM of ace- toxyroyleanone for 48 hours incubation 120 100 80 60 40 20 0 % G ro w th in hi bi tio n Concentration of AXR 5 µM 10 µM 20 µM 30 µM 40 µM THP-1 PC-3 T47D MCF-7 MiaPaCa-2 Colo-205 Caco-2 fR-2 Bez-235 (10 nm) + - - - AXR (µM) - 0 10 30 65 kDa 42 kDa 17 kDa Caspase-3 β-actin NF-kB (p 65) 144 Bangladesh J Pharmacol 2016; 11: 141-147 tion, progression and migration of numerous cancers (Bhart et al., 2003; Garg and Aggarwal, 2002). Notably, NF-κB promotes cell survival via the induction of pro- teins that inhibit components of the apoptotic machinery in normal and cancerous cells (Shen and Tergaonkar, 2009). To evaluate the mechanism by which the effect of the acetoxyroyleanone occurred, further experiments of the effect of acetoxyroyleanone on NF-κB protein expression was determined. The most abundant form of NF-κB consists of a p50 subunit and a p65 subunit. In its inactive form, NF-κB is located in the cytoplasm, however, upon activation by various stimuli, it translocates to the nucleus, where it may activate genes leading to cell survival or proliferation Figure 4: Acetoxyroyleanone treatment led significant decrease in NF-κB (p65) and increase in caspase-3 expression at 30 µM with respect to untreated and BEZ-235 (10 nm); ap≤0.05; bp≤0.01; cp≤0.001 Figure 5: Effect of acetoxyroyleanone on cell migration of Colo-205 monolayers. Cells were exposed to different concentrations of acetoxyroyleanone (10 and 30 µM) for 48 hours compared to untreated. A, represents BEZ-235 treatment which was used as positive control, B, C and D represent 0, 10 and 30 µM concentration of acetoxyroyleanone 18 16 14 12 10 8 6 4 2 0 % F ol d ch an ge (a ct in n or m al iz ed ) Bez-235 (10 nm) + - - - AXR (µM) - 0 10 30 NF-kB Caspase-3 a b c c c Bangladesh J Pharmacol 2016; 11: 141-147 145 http://www.ncbi.nlm.nih.gov/pubmed?term=Garg%20A%5BAuthor%5D&cauthor=true&cauthor_uid=12040437 http://www.ncbi.nlm.nih.gov/pubmed?term=Shen%20HM%5BAuthor%5D&cauthor=true&cauthor_uid=19212815 http://www.ncbi.nlm.nih.gov/pubmed?term=Tergaonkar%20V%5BAuthor%5D&cauthor=true&cauthor_uid=19212815 (Schmid et al., 2000; Birbach et al., 2002; Swinney et al., 2002). Notably, our study demonstrates that exposure to acetoxyroyleanone resulted in remarkable down- regulation in the expression of NF-κB (p65). Further- more, caspases play a central role in apoptosis and are central to the mechanism of apoptosis as they are both the initiators and executioners. Among caspases, caspase-3 is a frequently activated death protease, which catalyzes the specific cleavage of many key cellular proteins resulting apoptosis (Porter and Jänicke, 1999). Importantly, caspase-3 is crucial for apoptotic chromatin condensation and DNA fragmen- tation in all cell types. Here, it is reported that acetoxyroyleanone mediates caspase-3 up-regulation in colo-205 cells. Taken together, these data indicate that NF- κB and caspase-3 play a pivotal role in mediating acetoxyroyleanone-induced apoptosis in Colo-205 cells. Cell migration plays a critical role in tumor cell invasion and metastasis (Yamaguchi et al., 2005). Importantly, cell migration and invasion represents an important property for chemotherapeutic agent other than having the potential to cause specific cancer cell death. Molecules involved in cancer cell migration could be the potential target for anti-metastasis therapy. This study describes how colon cancer cell migrate using acetoxyroyleanone tested by measuring the gap between control and treatment groups. Acetoxyroyleanone was found actively inhibit colon cancer cell migration. Notably, apoptosis is morphologically characterized by chromatin condensation, internucleosomal fragments, cell shrinkage, membrane blebbing and formation of apoptotic bodies without disruption of the plasma membrane (Wyllie et al., 1984). Conclusion Acetoxyroyleanone has a target based antiproliferative activity. A successful anticancer drug must have the ability to induce tumor cell apoptosis and these results showed that acetoxyroyleanone does show this charac- teristic feature of inducing apoptosis in Colo-205 cancer cells. References Bhart AC, Donato N, Singh S, Aggarwal BB. 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Number of times you have read this paper 2. Quality of paper 3. Your comments Introduction: Conclusion: References: Text2: Dropdown3: [Click] Dropdown4: [0] DatePrinted: This article was downloaded by you on: Jun 27, 2017