ANTIBREAST CANCER ACTIVITY OF NANOPROPOLIS INDONESIA INDUCED MAMMARY GLAND TUMOR BY ON DMBA IN VIRGIN SPRAGUE-DAWLEY RATS AKHMAD ENDANG ZAINAL HASAN JUMALI1,2 2*, D MANGUNWIDJAJA , T C UNARTI , O SUPARNO A SETIYONOITI ANDRA S NO GUS2 2 3and 1 Institut Pertanian , Bogor 16680, IndonesiaDepartment of Biochemistry, Faculty of Mathematics and Sciences, Bogor 2 epartmen Engineering and TD t of Agroindustrial Technology, Faculty of Agricultural echnology, Institut Pertanian Bogor Bogor 16680, , Indonesia 3D t of Clinic, Reproduction and Pathology, Faculty of Veterinary, epartmen Institut Pertanian Bogor, Bogor 16680, Indonesia Received 15 March 2015/Accepted 15 May 2016 ABSTRACT The objective of this study was to determine the effect of nanopropolis induced rat mammary to cure cancer on tumor . After the first tumors appearance, twenty rats were using 7,12-dimethylbenz(a)anthracene (DMBA) eight divided into seven groups. Group 1, 2 and 3 served as , 32 and 56 recipient of nanopropolis dosages 8 µg/mL treatments served recipient of dosage µg/mL treatment served recipient of ; Group 4 as propolis of 233 ; Group 5 as do orubi in treatment served recipient of treatment (control) x c ; Group 6 as DMBA and Group 7 as normal group . The effect of nanopropolis of 32 µg m and propolis 233 µg m were similar in reducing umor dosage / L dosage of / L t size, healing the wounds caused by the tumor and It turns out that there is a relationship eliminating cancer cells. between particle size absorbent material . The study suggest that nanopropolis was very s ed with small concentration effective treat mammary gland tumors and breast cancersto rat . Keywords: breast cancer nanopropolis, propolis, prague-Dawley rat, S INTRODUCTION Cancer is one of the leading causes of death in the world, especially in developed countries and the second killer in developing countries. Based on data from the Hospital Information System (SIRS) in 2007 breast cancer ranks first until 2013, in hospitalized patients in all hospitals in Indonesia (16.85%), followed by cervical cancer (11.78%) (Ministry of Health 2013). In accordance with opinion of , tet alMuir . (2003) he cause of this disease a cover of estrogen-are dependent pathway, circulating androgen, estrogen and exposure to carcinogenic materials such as smoke, ultraviolet radiation, improper diet and stress; except for age and race that cannot be changed (Bates American Cancer Society 2016). In the early stage, breast cancer is only in the form of a lump in the mammary tissue and in an advanced stage resulting an injury to the mammary tissue. T this disease he ways to treat are the removal of tissue, radiation therapy and c h e m o t h e r a p y. R a d i a t i o n t h e r a p y a n d chemotherapy aim to destroy cancer cells are ed and control the disease so will the main tumor be shr nk, tumor growth will be slowed and u spread of cancer cells to other tissues will be prevented. Now been a lot of research esadays, there have to obtain cancer , either directly medications for the treatment of cancer to reduce theor side effects of chemotherapy. The use of medications such as doxorubicin is one to inhibit cancer mean growth and reduce the occurrence of new cancers. However, the negative impact of doxorubicin treatment is the occurrence of hair loss, heart rhythm disorders and decreased white blood cell count. There is a challenge to find cancer treatment is effective and minim which ize bad effects of the main treatment. ne effort is to O BIOTROPIA Vol. 23 No. 1, 2016: 35 - 41 35 DOI: 10.11598/btb.201 .2 . .6 3 1 473 * C orresponding author: , zainalhasan@ipb.ac.id pakzainalhasan@gmail.com mailto:zainalhasan@ipb.ac.id mailto:pakzainalhasan@gmail.com 36 find and develop from medication herbs and to do on herbal medication. Medication clinical test originated from herbs must be tested through a series of studies prior to its usage in cancer therapy. The tests should not involve animals. A b b a s a l i p o u r k a b i r . ( 2 0 1 0 ) a n d e t a l Purushothaman . (2012) investigated the et al incidence of breast cancer using virgin rats of Sprague-Dawley strain as test animals. In-vitro showed th studies at the origin of propolis locations in Indonesia from five and nanopropolis from Pandeglang ere le w ab to inhibit the growth of MCF-7 cell line. The nanopropolis inhibit the growth of MCF-7 cells ed at very low concentrations compared propolis to instead of nanoparticles . The (Hasan . 2014)et al purpose of this study was examineto the in-vivo antibreastcancer activity of nanopropolis on the DMBA induced of virgin Sprague-Dawley rats. MATERIALS AND METHODS Preparation of Nanopropolis Nanopropolis were prepared in three stages of homogenization using high speed homogenizer . Some modifications were done for preparing of nanopropolis particle as previously mentioned by Aimi . (2009), Bhaskar . (2009), Hasan . et al et al et al (2012), Chen (2006) and Kim . (2008)et al. et al . Mammary umor nductionT I Twenty eight rats were intraperitoneally injected using a m with ixture of DMBA ( concentration of /25 mg kgbody weight), olive oil and physiological saline. Experimental esignD After the first tumors appearance, twenty eight rats were divided into seven groups, with three animals each group. Group 1, 2 and 3 served asfor recipient of nanopropolis dosages of 8, 32 and 56 µg/mL treatments served ; Group 4 as recipient of dosage of 233 µg/mL propolis treatment served recipient of ; Group 5 as do orubi in treatment; served x c Group 6 as recipient of treatmentDMBA ; and Group 7 as (control)a normal group . Tumor sTwo dimen ional tumor areas were calculated as an ellips (Abbasalipourkabir . 2010). The e et al volume of tumor was calculated by the formula of: 2V = ab /2 w here a = the longest diameter = b the shortest diameter of tumor At sacrifice, the tumor were removed for histopathological . examination Hi topathology Analysiss siaAfter euthana , at the end of the study, the mammary tumor masses of rats were removed. Sample of tissues were fixed immediately in 10% formalin overnight, embedded in paraffin, cut into 4 m sections and stained with hematoxylin-μ eosin (HE). RESULTS AND DISCUSSION The results of body weight rats measurements after injection and treatment were presented in Figure 1. een in Figure 1 normal rat body As s , weight continued to increase with time , addition as well as other treatments. The DMBA treatment showed a decrease in body weight. According to Cordeiro d Kaliwal (2011), an effect of DMBA can reduce body weight rat caused by the of nature of its toxicity despite an increase in tumor volume. Tumor progress can be determined by measuring the volume test animal tumor of (Abbasalipourkabir 2010; Purushothaman et al. et al et al In the study of. 2012; Martic . 2011). Abbasalipourkabir . (2010) volume et al , tumor was calculated by measuring the length and width of the swelling, while et al.Purushotaman (2012) an calcu d the tumor et ald Martic . (2011) late volume by measuring the length, width and height of the tumor section. esults in mammary tissue R tumor volume of research data using the method of Abbasalipourkabir (2010) can be seen in et al. Figure een in Figure 2 increasing tumor 2. As s , volume in DMBA treatment happened only without propolis or nanopropolis treatments. BIOTROPIA Vol. 23 No. 1, 2016 Weeks after treatment Antibreast cancer n Akhmad E Z H – et al. activity of nanopropolis Indonesia o induced mammary . . 37 The propolis or nanopropolis treatments performed in rats decreased tumor volume. Even in the 32 and 56 nanopropolis µg/mL treatment and 233 µg/mL propolis treatment there were decline after an increase in tumor volume in the fi th week after being given treatment. When f viewed from the point of decline, the injection of 56 µg/mL treatment hadnanopropolis sharp corners the tumor size reduction process , o red rccur very quickly. This occur ence may be due to the relatively high dose of the 50(6 x IC ) ingredients in nanopropolis sufficient to that is eliminate cancer cells. Influence the amount of of the active ingredient component in nano- propolis ) was in trumental in (32 and 56 µg/mL s comparison with the influence of the size particle of the repair the component nanopropolis for tissue from tumor to compare propolis at 56 µg/mL. N a n o p r o p o l i s t r e a t m e n t w i t h t h e concentration 8 ha been done to heal of µg/mL d cell and tissue, although there are tumor to repair Figure 1 dy weight of rats after induction of DMBA and prior necropsy (1 = group nanopropolis 8 µg, 2 = Bo s of group of nanopropolis 32 µg, 3 = group nanopropolis 56 µg, 4 = group propolis µg, 5s s of s of 233 = doxorubicin s of group , 6 = group DMBA and 7 = normal control Weeks after treatment Figure 2 Tumor volume of mice after induction by DMBA prior to necropsy (1 = group nanopropolis 8 µg , of /mL 2 = group of nanopropolis 32 µg , 3 = group nanopropolis 56 µ , 4 = group propolis /mL of g/mL of 233 /mL doxorubicin of group µg , 5 = group, 6 = group DMBA and 7 = normal control ) 38 BIOTROPIA Vol. 23 No. 1, 2016 cells. On tre tment with a concentration of 32 a µg/mL, had occurredformation of intact tissue to washeal and subcutaneous fat formed and there were e rpr sence of intact epide mal glands and many gl bular epithelium as well as o s reversal of in ,tact tumor draining wounds. Similarly nanopropolis treatment at a concentration 56 of µg/mL r to lower occu red heal much better than concentration of result nanopropolis. This show thated the amount of active ingredient content affected more to the healing rate of cancer. Figure 3 showed the result of administration of nanopropolis on rat mammary tissue (32 and 56 ) which ha repair skin µg/mL d ed tissue after induced by DMBA being . T issue conditions were better in he t nanopropolis concentration of Fig 456 µg/mL ( . ), the formation of new tissue was much more than giving nanopropolis at concentration of and 8 32 µg/mL. d of This prove that the administration nanopropolis increasingly play a role in ed mammary tissue wounds caused by DMBA induced tumor results. This result showed that the amount of active ingredient affected the healing rate of cancer. The greater the concentration nanopropolis used, the greater of the effect tumor tissue healing wounds. The on influence of different concentrations and time of administration of propolis against different cancer cells ha also been studied by d Bufalo . et al (2007). Presence of a tumor as a result of wound healing propolis administration s the using wa formation of epithelial tissues been that had studied by de Moura . (2011), although using et al different propolis. This condition was supported by the presence of components of propolis (Hasan . 2014) or mineral contents (Hasanet al et al. 2013). At a concentration of the 56 µg/mL mammary were healthy and aesthetically alveoli clean In Figure 4a seen the contain . it is ingalveoli blood plasma (blue arrows pointing as result of ) a mammary tissue repair. On other rats the groups (DMBA or negative control) in which group nanopropolis or propolis injection was not p , erformed mammary tissue condition was still going on angiogenesis and cancer cells enters blood vessels. In this condition, cancer cell Figure 3 The mammary tissue of virgin rat after induced by MBA and received treatment of being D injection a. nanopropolis (32 µg/mL) b. nanopropolis (56 µg/mL) ( = skin epi = and blue arrow thelium, yellow arrow normal hair licles white arrow capillary epidermis HE stainingfol , = ) ( , 200x) Figure 4 a) The ammary tissue of rats SD induced by DMBA and nanopropolis every seven days within m 56 mg/mL two months, b) The mammary tissue of rats SD induced by DMBA and no treatment within two months after induction (black arrow blue arrow = filled blood plasma white arrow = cancer cells, = , with , alveole alveole red arrow = connective tissue) HE staining, ( 200x) 39 spread in so much connective tissue and almost eve y cancer cells r tissue as is shown in Figure 4b ( (white arrows) and connective tissue (red arrows) .) The physical condition of the mammary tissue healing drying can be seen when condition occur in an area that has suffered injury due to red the tumor Fig 5a While the mammary tissue ( . ). was swollen , caused by the tumor the effect of DMBA induced in virgin rats were large swell (Fig. 5b). Even one rat in this group had inflammation of the mammary tissue. his condition can be T refe red to as stage IV breast cancerr . Tested activities of propolis curing cancer proved dosage 233 µg/mL c that a of an heal damage by the tumor tissue compared s caused with the positive control treatment without induction by DMBA treatment. Positive control (without induction by DMBA) does not cure cancer develop accumulation of and even ed cancer cells and tissue damage Fig 6( . ). Inoue et al. (2008) showed that the inhibition of cancer growth occur ed after concentration of r propolis 320 µg/mL. , Bermúdez (2006) Similarly et al. reported that 10%wound healing with propolis only occur as much as only the 60% for occurrence . In breast of re-epithelialization - tissue repair seen around the damaged can be tissue with (blue arrow) and re-epithelialization hair l ( For the follic es yellow arrow) in Figure 6a. DMBA research, induced mammary wounds were formed in the mammary tissue and there were many cancer cells Fig ( . 6b). F shows drying igure 7 cancer cells condition with black spots on the new blood vessels, but there are still cancer cells expected to remain active. This is because the treatment required quite long , so that the healing process a time was still running and still not finished On was . treatment with propolis 233 µgdosage of /mL, the drying process indicated the healing in tissues affected by cancer. dosage µg/mL At of 32 nanopropolis has the ability to cure cancer which to has the same effect a s mu ch as 2 3 3 o f p r o p o l is µ g/ m L administration. The particle size in nanopropolis is very small so active ingredient remaining in , nanopropolis can log into the network with ease, while the of propolis with 233 dosage µg/mL which was active against tumor growth is caused Figure 5 a) The physical condition of the mammary tissue healing can be experienced with drying occur in an area that has s suffered injury due to tumor after injection of b) non treatment or only induction of nanopropolis 32 µg/mL, DMBA c) for ( = , and condition before treatment after induction of DMBA 90 days yellow arrow dry wound, white arrow mammary swelling bec use of tumor = a ) Figure 6 rat mammary tumor tissue after being induced by DMBA and treated with injection of (a) propolis (233 Virgin μg/mL) and (b) Without treatment (positive control) (blue arrow = skin epithelium, yellow arrow = normal hair follicle, white arrow = inflammation) (HE staining, 200x) Antibreast cancer n Akhmad E Z H – et al. activity of nanopropolis Indonesia o induced mammary . . 40 BIOTROPIA Vol. 23 No. 1, 2016 by the presence of the active components in propolis prevent tumor progression. This fact ing was caused by the presence of compounds in either nanopropolis or propolis, such as organic acids like firulic acid and acid, polyphenols caffeic and flavonoids in propolis inhibit the which proliferation of cancer cells ole of . The r flavonoids and ca fe acid is to inhibit the f ic formation of protein kinases that are used for cell proliferation result is going cell which to inhibit formation process and apoptosis induced occur (Madeo . 2004). In accordance rence et al with the results of de Moura (2011), the et al. healing of wounds caused by the tumor may occur due to administration of propolis. Meanwhile, according to Sun . (2012) crysin components et al present in propolis can decrease the volume of tumor that occurs in the mammary tissue of DMBA induced . The research conducted by mice Bhattacharjee . (2012) and Lim . (2011) et al et al states that there is a relationship between particle size absorbent materials on the healing of cancer. CONCLUSIONS The effect of nanopropolis of 32 s with dosage µ /mL with dosage of µ /mL g and propolis 233 g w the same in ere reducing tumor size, healing the wounds caused by the tumor and eliminat ing cancer cells. It turn out that there is a ed relationship between particle ize absorbent s material . The study s on the healing of cancer sug gest that nanopropolis ed with small concentration (dosage of 32 µg/mL) was very effective for the treatment of rat mammary gland tumors and breast cancers. ACKNOWLEDGEMENTS The authors to thank BPPS Fellowship wish of Indonesian Ministry of Education and Culture, and SEAMEO BIOTROP for their financial supports and Institut Pertanian Bogor for its facilities for conducting this study. REFERENCES Abbasalipourkabir R, Salehzadeh A, Abdullah R. 2010. Antitumor activity of tamoxifen loaded solid lipid nanoparticles on induced mammary tumor gland in Sprague-Dawley rats. Afr J Biotech 9(43):7337- 45. Aimi M, Nemori R, Ogiwara K .. 2009. Casien Nanoparticle Inventor. Bates American Cancer Society [Internet]. 2016. Causes, Risk Factors, and Prevention Topics. USA: American Cancer Society; [cited 2016 May 31]. Available from: http://www.cancer.org/cancer/ breastcancer/detailedguide/breast-cancer-risk- factors. Bermúdez IC, García GS, Piloto AA, Pérez YF, Valdivieso AG. 2006. Effect of the Cuban propolis collected in Manzanillo area on the wounds healing in rats. Pharmacol 3:416-21. Bhaskar K, Anbu , Ravichandiran , Venkateswarlu , Rao J V V YM. 2009. Lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, in-vitro, ex-vivo and studies. Lipids Health Disease 8(6):758-in vivo 76. Bhattacharjee S, Erchov D, Fytianos K, van der Gucht J, Alink GM, Rietjens IMCM, Marcelis ATM, Zuilhof H. 2012. Cytotoxicity and cellular uptake of triblock copolymer nanoparticles with different size and surface characteristics. Particle Fibre Toxicol 9(11):1-19. Figure 7 Mammary tissue (a) and skin tissue (b) DMBA-induced SD rats treated with propolis at a dosage of 233 g/mL µ every 7 days within 2 months (red arrows = vein, white arrow = dead cancer cells, black arrow = hair follicle) (HE staining, 200x) http://www.cancer.org/cancer/ 41 Bufalo MC, Candeias MGJ, Sforcin JM. 2007. In vitro cytotoxic effect of Brazillian green propolis on human laryngeal epidemoid carcinoma (HEP-2) cells. Advance Access Pub 6(4):483-7. Chen M, Diao G, Zhang E. 2006. Study of inclusion complex of beta-cyclodextrin and nitrobenzene. Chem 63:522-9. Cordeiro MC, Kaliwal BB. 2011. Antioxidant activity of bark extract if SPRENG on DMBA Bridelia retusa induced mammary carcinogenesis in female sprague Dawley rats. J Ph rm 2(1):14-20.a de Moura SAL, Negri G, Salatino A, Lima LDC, Dourado LPA, Mendes JB, Andrade SP, Ferreira MAND, Cara DC. 2011. Aqueous extract of Brazilian green propolis: primary components, evaluation of inflammation and wound healing by using subcutaneous implanted sponges. Evid Based Complement Alternat Med 2011:1-8. Hasan AEZ, Ambarsari L, Artika IM, Julistiono H, Tarunasari D. 2013. Induction resistance of Candida sp. Y390 to ethanol stress by kopyor coconut and virgin coconut oil. Emir J Food Agric 25(10):790-7. Hasan AEZ, Artika IM, Fahri VR, Sari N. 2012. Potency of nanopropolis from stingless bee spp. as Trigona antibacteria gents. Chem Progress 5(1):1-7.a Hasan AEZ, Mangunwidjaja D, Sunarti TC, Suparno O, Setiyono A. 2014. Investigating the antioxidant and anticytotoxic activities of propolis collected from five regions of Indonesia and their abilities to induce apoptosis. Emir J Food Agric 26(5):390-8. Inoue K, Saito M, Kanai T, Kawata T, Shigematsu N, Uno T, Isobe K, Liu CH, Ito H. 2008. Antitumor effects of water soluble propolis on a mouse sarcoma cell line and . Am J Chin Med 36(3):625-34.in vivo in vitro Kim DM, Lee , Aum , Kim 2008. Preparation of GD SH HJ. propolis nanofood and application to human cancer. Biol Pharm Bull 31(9):1704 10. - Lim HN, Nurzulaikha R, Harrison I, Lim SS, Tan WT, Yeo MC. 2011. Spherical tin oxide, SnO particles 2 fabricated via facile hydrothermal method for detection of mercury (II) ions. Int J Electrochem Sci 6:4329-40. Madeo F, Herker , Wissing , Jungwirth , Eisenber , E S H T Frohlich . 2004. Apoptosis in yeast. Current KU Opinion Microbiol 7:655–60. Martic K, Vlacic Z, Rudman F, Lambasa S, Tomasovic- Loncaric C, Stanec Z. Tumor and reast 2011. b volume ratio as a predictive factor for axillary lymph node metastases in T1c ductal invasive breast cancer: prospective obser vational clinico- pathological study. Jpn J Clin Oncol 41(12):1322-6. Ministry of Health . 2013. [Internet] Indonesia: Ministry of Health; [cited 2013 June 14]. Available from: http://www.depkes.go.id/index.php/berita/press- release/2233-seminar-sehari -dalam-rangka- memperingati-hari-kanker-sedunia-2013.html. Muir D, Kanthan R, Kanthan SC. 2003. Male versus female breast cancers. Arch Pathol Lab Med 127(1):36-41. Purushothaman A, Nandhakumar E, Sachdananram P. 201 . Anticancer effect of shemamrithaa (a 2 phytochemical formulation) on 7,12-dimethyl benz(a)anthracene induced mammary carcinoma in rats. Asian J Pharm Clinical Res 5(1):101-7 . Sun LP, Chen AL, Hung AC, Chien YH, Huang JS, Huang CY, Chen YW, Chen CN. 2012. Chrysin: a histone deacetylase 8 inhibitor with anticancer activity and a suitable candidate for the standardization of Chinese propolis. J Agric Food Chem 60:11748-58. Antibreast cancer n Akhmad E Z H – et al. activity of nanopropolis Indonesia o induced mammary . . http://www.depkes.go.id/index.php/berita/press-