Abstract: Objective: Fruit of Phyllanthus emblica Linn. (PE) is widely consumed as a functional food and used as a folk medicine due to its remarkable nutritional and pharmacological effects. Mitomycin C (MMC) and cisplatin (cDDP) are the most widely used forms of chemotherapeutic drug, but their clinical use is limited by their genotoxicity to normal cells. We aimed to determine whether PE has potential to reduce the genotoxicity, while improving the anticancer effect, of MMC and cDDP. Methods: Cell proliferation was evaluated using the trypan blue exclusion assay and colony-forming assay. Genomic instability (GIN) was measured using the cytokinesis-block micronucleus assay. Results: Co-treatment (72 h) with PE at 20–320 μg/ml significantly enhanced the efficacy of MMC (0.05 μg/ml) and cDDP (1 μg/ml) against Colo205 colorectal cancer cells (P<0.05), and at 80–320 μg/ml significantly decreased MMC- and cDDP-induced GIN and multinucleation in normal colonic NCM460 cells (P<0.05). PE significantly decreased the mitotic index (P<0.01), blocked mitotic progression (P<0.05), and promoted apoptosis (P<0.01) in MMC- and cDDP-treated NCM460 cells, suggesting that PE-mediated inhibition of mitosis and induction of apoptosis may limit the division and survival of highly damaged cells. Also, PE was found to inhibit the clonal expansion of MMC- and cDDP-treated NCM460 cells (P<0.05) and decrease the heterogeneity of the surviving clones. Conclusions: PE potentiates the anticancer efficacy of MMC and cDDP, while preventing their genotoxicity and inhibiting clonal expansions of unstable genomes in normal cells. These data suggest that PE has the potential to reduce the risk of secondary cancers induced by chemotherapeutics.

余甘子提取物增强丝裂霉素C和顺铂的抗癌作用并降低其对正常细胞的遗传毒性

[1]Aiyer, H.S., Vadhanam, M.V., Stoyanova, R., et al., 2008. Dietary berries and ellagic acid prevent oxidative DNA damage and modulate expression of DNA repair genes. Int. J. Mol. Sci., 9(3):327-341.

[2]Aronson, J.K., 2010. Meyler’s Side Effects of Drugs Used in Cancer & Immunology. Elsevier, Amsterdam.

[3]Attia, S.M., 2010. The impact of quercetin on cisplatin induced clastogenesis and apoptosis in murine marrow cells. Mutagenesis, 25(3):281-288.

[4]Banu, S.M., Selvendiran, K., Singh, J.P.V., et al., 2004. Protective effect of Emblica officinalis ethanolic extract against 7,12-dimethylbenz(a)anthracene (DMBA) induced genotoxicity in Swiss albino mice. Hum. Exp. Toxicol., 23(11):527-531.

[5]Brüsehafer, K., Rees, B.J., Manshian, B.B., et al., 2014. Chromosome breakage induced by the genotoxic agents mitomycin C and cytosine arabinoside is concentration and p53 dependent. Toxicol. Sci., 140(1):94-102.

[6]Cheung-Ong, K., Giaever, G., Nislow, C., 2013. DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology. Chem. Biol., 20(5):648-659.

[7]De, A., De, A., Papasian, C., et al., 2013. Emblica officinalis extract induces autophagy and inhibits human ovarian cancer cell proliferation, angiogenesis, growth of mouse xenograft tumors. PLoS ONE, 8(8):e72748.

[8]Deans, A.J., West, S.C., 2011. DNA interstrand crosslink repair and cancer. Nat. Rev. Cancer, 11(7):467-480.

[9]Dertinger, S.D., Avlasevich, S.L., Torous, D.K., et al., 2014. Persistence of cisplatin-induced mutagenicity in hematopoietic stem cells: implications for secondary cancer risk following chemotherapy. Toxicol. Sci., 140(2):307-314.

[10]Eng, C., 2010. Are herbal medicines ripe for the cancer clinic? Sci. Transl. Med., 2(45):45ps41.

[11]Fenech, M., 2006. Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability, mitotic dysfunction and cell death. Mutat. Res. Fundam. Mol. Mech. Mutagen., 600(1):58-66.

[12]Fenech, M., 2007. Cytokinesis-block micronucleus cytome assay. Nat. Protoc., 2(5):1084-1104.

[13]Fenech, M., Chang, W.P., Kirsch-Volders, M., et al., 2003. HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat. Res. Genet. Toxicol. Environ. Mutagen., 534(1-2):65-75.

[14]Fenech, M., Kirsch-Volders, M., Natarajan, A., et al., 2011. Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis, 26(1):125-132.

[15]Giri, A., Khynriam, D., Prasad, S.B., 1998. Vitamin C mediated protection on cisplatin induced mutagenicity in mice. Mutat. Res., 421(2):139-148.

[16]Glen, C.D., Dubrova, Y.E., 2012. Exposure to anticancer drugs can result in transgenerational genomic instability in mice. Proc. Natl. Acad. Sci. USA, 109(8):2984-2988.

[17]Greaves, M., Maley, C.C., 2012. Clonal evolution in cancer. Nature, 481(7381):306-313.

[18]Guo, X., Wang, X., 2016. Phyllanthus emblica fruit extract activates spindle assembly checkpoint, prevents mitotic aberrations and genomic instability in human colon epithelial NCM460 cells. Int. J. Mol. Sci., 17(9):1437.

[19]Guo, X., Ni, J., Liu, X., et al., 2013. Phyllanthus emblica L. fruit extract induces chromosomal instability and suppresses necrosis in human colon cancer cells. Int. J. Vitam. Nutr. Res., 83(5):271-280.

[20]Guo, X., Ni, J., Xue, J., et al., 2017. Extract of bulbus Fritillaria cirrhosa perturbs spindle assembly checkpoint, induces mitotic aberrations and genomic instability in human colon epithelial cell line. Exp. Toxicol. Pathol., 69(3):163-171.

[21]Heng, H.H., Bremer, S.W., Stevens, J.B., et al., 2013. Chromosomal instability (CIN): what it is and why it is crucial to cancer evolution. Cancer Metast. Rev., 32(3-4):325-340.

[22]Iamsaard, S., Arun, S., Burawat, J., et al., 2014. Phenolic contents and antioxidant capacities of Thai-Makham Pom (Phyllanthus emblica L.) aqueous extracts. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 15(4):405-408.

[23]Janssen, A., Kops, G.J., Medema, R.H., 2009. Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells. Proc. Natl. Acad. Sci. USA, 106(45):19108-19113.

[24]Kempf, S.R., Ivankovic, S., 1986. Carcinogenic effect of cisplatin (cis-diammine-dichloroplatinum (II), CDDP) in BD IX rats. J. Cancer Res. Clin. Oncol., 111(2):133-136.

[25]Lam, W., Bussom, S., Guan, F., et al., 2010. The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci. Transl. Med., 2(45):45ra59.

[26]Lengauer, C., Kinzler, K.W., Vogelstein, B., 1997. Genetic instability in colorectal cancers. Nature, 386(6625):623-627.

[27]Li, F.Y., Lai, M.D., 2009. Colorectal cancer, one entity or three. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 10(3):219-229.

[28]Li, X., Yang, G., Li, X., et al., 2013. Traditional Chinese medicine in cancer care: a review of controlled clinical studies published in Chinese. PLoS ONE, 8(4):e60338.

[29]Luo, W., Zhao, M., Yang, B., et al., 2011. Antioxidant and antiproliferative capacities of phenolics purified from Phyllanthus emblica L. fruit. Food Chem., 125(2):277-282.

[30]Maley, C.C., Galipeau, P.C., Li, X., et al., 2004. The combination of genetic instability and clonal expansion predicts progression to esophageal adenocarcinoma. Cancer Res., 64(20):7629-7633.

[31]Mathai, R.T., Tonse, R., Kalekhan, F., et al., 2015. Amla in the prevention of aging: scientific validation of the ethnomedicinal claims. In: Watson, R.R. (Ed.), Foods and Dietary Supplements in the Prevention and Treatment of Disease in Older Adults. Academic Press, Waltham, p.29-35.

[32]Mazumdar, M., Giri, S., Giri, A., 2011. Role of quercetin on mitomycin C induced genotoxicity: analysis of micronucleus and chromosome aberrations in vivo. Mutat. Res. Genet. Toxicol. Environ. Mutagen., 721(2):147-152.

[33]Meadows, A.T., Friedman, D.L., Neglia, J.P., et al., 2009. Second neoplasms in survivors of childhood cancer: findings from the childhood cancer survivor study cohort. J. Clin. Oncol., 27(14):2356-2362.

[34]Min, K., Ebeler, S.E., 2009. Quercetin inhibits hydrogen peroxide-induced DNA damage and enhances DNA repair in Caco-2 cells. Food Chem. Toxicol., 47(11):2716-2722.

[35]Mirunalini, S., Krishnaveni, M., 2010. Therapeutic potential of Phyllanthus emblica (AMLA): the ayurvedic wonder. J. Basic Clin. Physiol. Pharmacol., 21(1):93-105.

[36]Moyer, M.P., Manzano, L.A., Merriman, R.L., et al., 1996. NCM460, a normal human colon mucosal epithelial cell line. In Vitro Cell. Dev. Biol. Anim., 32(6):315-317.

[37]Pan, S.S., Andrews, P.A., Glover, C.J., et al., 1984. Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase. J. Biol. Chem., 259(2):959-966.

[38]Pinmai, K., Chunlaratthanabhorn, S., Ngamkitidechakul, C., et al., 2008. Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents: doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells. World J. Gastroenterol., 14(10):1491-1497.

[39]Ruan, W.J., Lai, M.D., Zhou, J.G., 2006. Anticancer effects of Chinese herbal medicine, science or myth? J. Zhejiang Univ.-Sci. B, 7(12):1006-1014.

[40]Sharma, N., Trikha, P., Athar, M., et al., 2000. Inhibitory effect of Emblica officinals on the in vivo clastogenicity of benzo alpyrene and acyclophosphamide in mice. Hum. Exp. Toxicol., 19(6):377-384.

[41]Storchova, Z., Kuffer, C., 2008. The consequences of tetraploidy and aneuploidy. J. Cell Sci., 121(23):3859-3866.

[42]Torre, L.A., Bray, F., Siegel, R.L., et al., 2015. Global cancer statistics, 2012. CA Cancer J. Clin., 65(2):87-108.

[43]Travis, L.B., Holowaty, E.J., Bergfeldt, K., et al., 1999. Risk of leukemia after platinum-based chemotherapy for ovarian cancer. New. Eng. J. Med., 340(5):351-357.

[44]van den Belt-Dusebout, A.W., Wit, R.D., Gietema, J.A., et al., 2007. Treatment-specific risks of second malignancies and cardiovascular disease in 5-year survivors of testicular cancer. J. Clin. Oncol., 25(28):4370-4378.

[45]Waters, M.D., Brady, A.L., Stack, H.F., et al., 1990. Antimutagenicity profiles for some model compounds. Mutat. Res. Rev. Genet. Toxicol., 238(1):57-85.

[46]Zaki, B.I., Suriawinata, A.A., Eastman, A.R., et al., 2014. Chromosomal instability portends superior response of rectal adenocarcinoma to chemoradiation therapy. Cancer, 120(11):1733-1742.