In vitro assessment of cytotoxic, apoptotic and genotoxic effects of metformin

Yıl 2021, Cilt: 51 Sayı: 2, 167 - 174, 31.08.2021

Tuba Sarıaydın Tuğbagül Çal Sevtap Aydın Dilsiz Hande Canpınar Ülkü Ündeğer Bucurgat

Öz

Background and Aims: Recent studies have shown the anticancer properties of metformin, which is widely used in diabetes mellitus. The possible mechanisms of anticancer effects of metformin have not been fully elucidated. We aimed to investigate the cytotoxic, genotoxic, and apoptotic effects of metformin in HepG2 and HeLa cells. Methods: The cytotoxicity, genotoxicity, and apoptotoic effects were determined by MTT method, Comet assay, and FACS assay, respectively. Results: Metformin significantly decreased cell viability above 4 and 32 mM in HepG2 and HeLa cells, respectively, for 48 h. The IC50 values were 57.3 mM (HepG2) and 76.9 mM (HeLa). Metformin (5-1000 μM) alone did not increase DNA damage in all cells. It did not change oxidative DNA damage in HepG2 cells but induced oxidative DNA damage in HeLa cells. HepG2 cells treated with only 32 mM metformin revealed 10% apoptosis. G0/G1 phase accumulation was statistically higher in the cells treated with 4, 8, and 64 mM metformin (91%, 99%, and 97% respectively) than in (-) control (80%). HeLa cells revealed apoptosis of 30%, 39%, 27% at 4, 32, and 64 mM concentrations, respectively. The results implicate that the inhibition of HepG2 cell viability may be due to the arrest of cell cycle in G0/G1 phase and apoptosis, whereas apoptotic response is mainly responsible for the cytotoxicity of metformin in HeLa cells. Conclusion: Metformin may not induce DNA damage at non-cytotoxic high doses and lead to apoptosis, even if compatible with previous data. This study provides important information that metformin may play an essential role in apoptosis and cell cycle progression in carcinoma cell lines, which can explain the anticancer effects of metformin, but further studies are needed to support these results.

Anahtar Kelimeler

Metformin, genotoxicity, apoptosis, HepG2 cells, HeLa cells

Destekleyen Kurum

Hacettepe University Scientific Research Projects Coordination Unit

Proje Numarası

THD-2017- 13153

Kaynakça

• • Algire, C., Moiseeva, O., Deschênes-Simard, X., Amrein, L., Petruccelli, L., Birman, E., Viollet, B., Ferbeyre, G. & Pollak, M. N. (2012). Metformin reduces endogenous reactive oxygen species and associated DNA damage. Cancer Prevention Research, 5(4), 536-543.
• • Amador, R. R., Longo, J. P. F., Lacava, Z.G., Dórea, J. G. & Almeida Santos, M. F. M. (2012). Metformin (dimethyl-biguanide) induced DNA damage in mammalian cells. Genetics and Molecular Biology, 35(1), 153-158.
• • Attia, S. M., Helal, G. K., & Alhaider, A. A. (2009). Assessment of genomic instability in mormal and diabetic rats treated with metformin. Chemico-Biological Interactions, 180(2), 296-304.
• • Ben Sahra, I., Laurent, K., Loubat, A., Giorgetti-Peraldi, S., Colosetti, P., Auberger, P., Tanti, J. F., Le Marchand-Brustel, Y., & Bost, F. (2008). The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene, 27, 3576-3586.
• • Cai, X., Hu, X., Cai, B., Wang, Q., Li, Y., Tan, X. … Jing, X. (2013). Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo. Oncology Reports, 30(5), 2449-2457.
• • Cai, X., Hu, X., Tan, X., Cheng, W., Wang, Q., Chen, X., Guan, Y., Chen, C., & Jing, X. (2015). Metformin Induced AMPK Activation, G0/G1 Phase Cell Cycle Arrest and the Inhibition of Growth of Esophageal Squamous Cell Carcinomas In Vitro and In Vivo. PLoS One, 10(7), e0133349.
• • Colquhoun, A. J., Venier, N. A., Vandersluis, A. D., Besla, R., Sugar, L. M., Kiss, A. …Venkateswaran, V. (2012). Metformin enhances the antiproliferative and apoptotic effect of bicalutamide in prostate cancer. Prostate Cancer and Prostatic Diseases, 15(4), 346-352.
• • Dombrowski, F., Mathieu, C., & Evert, M. (2006). Hepatocellular neoplasms induced by low-number pancreatic islet transplants in autoimmune diabetic BB/Pfd rats. Cancer Research, 66(3), 1833- 1843.
• • Donadon, V., Balbi, M., Casarin, P., Vario, A., & Alberti, A. (2008). Association between hepatocellular carcinoma and type 2 diabetes mellitus in Italy: Potential role of insulin. World Journal of Gastroenterology, 14(37), 5695-5700.
• • Giovannucci, E. & Michaud, D. (2007). The role of obesity and related metabolic disturbances in cancers of the colon, prostate, and pancreas. Gastroenterology, 132(6), 2208-2225.
• • Jalving, M., Gietema, J. A., Lefrandt, J. D., de Jong, S., Reyners, A. K. L., Gans, R. O. B., & de Vries, E. G. E. (2010). Metformin: taking away the candy for cancer? European Journal of Cancer, 46(13), 2369-2380.
• • Janjetovic, K., Harhaji-Trajkovic, L., Misirkic-Marjanovic, M., Vucicevic, L., Stevanovic, D., Zogovic, N., Sumarac-Dumanovic, M., Micic, D., & Trajkovic, V. (2011). In vitro and in vivo anti-melanoma action of metformin. European Journal of Pharmacology, 668, 373- 382.
• • Kamarudin, M. N. A., Sarker, M. M. R., Zhou, J. R., & Parhar, I. (2019). Metformin in colorectal cancer: molecular mechanism, preclinical and clinical aspects. Journal of Experimental & Clinical Cancer Research, 38(1), 491-514.
• • Kane, D. A., Andersen, E. J., Price, J. W., Woodlief, T. L., Lin, C. T., Bikman, B. T., Cortright, R. N., & Neufer, P. D. (2010). Metformin selectively attenuates mitochondrial H2O2 emission without affecting respiratory capacity in skeletal muscle of obese rats. Free Radical Biology & Medicine, 49, 1082-1087.
• • Kefas, B. A., Cai, Y., Kerckhofs, K., Ling, Z., Marten, G., Heimberg, H., Pipeleers, D., & de Casteele, M. V. (2004). Metformin induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochemical Pharmacology, 68, 409-416.,
• • Kheirandish, M., Mahboobi, H., Yazdanparast, M., Kamal, W., & Kamal, M.A. (2018). Anti-cancer effects of metformin: Recent evidences for its role in prevention and treatment of cancer. Current Drug Metabolism, 19(9), 793-797.
• • Kim, M.Y., Kim, Y.S., Kim, M., Choi, M.Y., Roh, G.S., Lee, D.H. … Choi, W.S. (2019). Metformin inhibits cervical cancer cell proliferation via decreased AMPK O-GlcNAcylation. Animal Cells and Systems, 23(4), 302-309.
• • Kobayashi, M., Kato, K., Iwama, H., Fujihara S., Nishiyama, N., Mimura, S. …Masaki, T. (2013). Antitumor effect of metformin in esophageal cancer: in vitro study. International Journal of Oncology, 42(2), 517-24.
• • Leone, A., Di Gennaro, E., Bruzzese, F., Avallone, A., & Budillon, A. (2014). New perspective for an old antidiabetic drug: Metformin as anticancer agent. Cancer Treatment and Research, 159, 355-376.
• • Lopez-Bonet E, Buxó M, Cuyàs E, Pernas, S., Dorca, J., Álvarez, I. … Menendez J.A. (2019). Neoadjuvant metformin added to systemic therapy decreases the proliferative capacity of residual breast cancer. Journal of Clinical Medicine, 8(12), 2180-2197.
• • Na, H. J., Park, J. S., Pyo, J.H., Lee, S. H., Jeon, H. J., Kim, Y. S., & Yoo, M. A. (2013). Mechanism of metformin: Inhibition of DNA damage and proliferative activity in Drosophila midgut stem cell. Mechanisms of Ageing and Development, 134(9), 381-390.
• • Onaran, I., Guven, G. S., Ozdas, S. B., Kanigur, G., & Vehid, S. (2006). Metformin does not prevent DNA damage in lymphocytes despite its antioxidant properties against cumene hydro-peroxideinduced oxidative stress. Mutation Research, 611, 1-8.
• • Ouslimani, N., Peynet, J., Bonnefont-Rousselot, D., Therond, P., Legrand, A., & Beaudeux, J. L. (2005). Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells. Metabolism, 54, 829-834.
• • Piro, S., Rabuazzo, A. M., Renis, M. & Purello, F. (2012). Effects of metformin on oxidative stress, adenine nucleotides balance and glucose-induced insulin release impaired by chronic FFA exposure in rat pancreatic islets. Journal of Endocrinological Investigation, 35(5), 504-510.
• • Saito, T., Chiba, T., Yuki, K., Zen, Y., Oshima, M., Koide, S. …Yokosuka, O. (2013). Metformin, a diabetes drug, eliminates tumor-initiating hepatocellular carcinoma cells. PloS One. 8(7), e70010.
• • Salani, B., Del Rio, A., Marini, C., Sambuceti, G., Cordera, R., & Maggi, D. (2014). Metformin, cancer and glucose metabolism. Endocrine- Related Cancer, 21(6), 461-471.
• • Siegel, R. L., Miller, K. D., & Jemal, A. (2019). Cancer statistics. CA: a Cancer Journal for Clinicians, 69(1), 7-34.
• • Sun, Y., Tao, C., Huang, X., He, H., Shi, H., Zhang, Q., & Wu, H. (2016). Metformin induces apoptosis of human hepatocellular carcinoma HepG2 cells by activating an AMPK/p53/miR23a/FOXA1 pathway. OncoTargets and Therapy, 9, 2845-2853.
• • Tyszka-Czochara, M., Bukowska-Strakova, K., & Majka, M. (2017). Metformin and caffeic acid regulate metabolic reprogramming in human cervical carcinoma SiHa/HTB35 cells and augment anticancer activity of Cisplatin via cell cycle regulation. Food and Chemical Toxicology, 106, 260-272.
• • Tyszka-Czochara, M., Konieczny, P., & Majka, M. (2017). Caffeic acid expands antitumor effect of metformin in human metastatic cervical carcinoma HTB-34 cells: implications of AMPK activation and impairment of fatty acids de novo biosynthesis. International Journal of Molecular Sciences, 18(2), 462-478.
• • Wang, Y., Xu, W., Yan, Z., Zhao, W., Mi, J., Li, J., & Yan, H. (2018). Metformin induces autophagy and G0/G1 phase cell cycle arrest in myeloma by targeting the AMPK/mTORC1 and mTORC2 pathways. Journal of Experimental & Clinical Cancer Research, 37(1), 63.
• • Will, M.A., Palaniappan, M., Peegel, H., Kayampilly, P., & Menon, K.M. (2012). Metformin: Direct inhibition of rat ovarian theca-interstitial cell proliferation. Fertility and Sterility, 98(1), 207-214.
• • Xia, C., Chen, R., Chen, J., Qi, Q., Pan, Y., Du, L., Xiao, G., & Jiang, S. (2017). Combining metformin and nelfinavir exhibits synergistic effects against the growth of human cervical cancer cells and xenograft in nude mice. Scientific Reports, 7, 43373.
• • Xia, C., Liu, C., He, Z., Cai, Y. & Chen, J. (2020). Metformin inhibits cervical cancer cell proliferation by modulating PI3K/Aktinduced major histocompatibility complex class I-related chain A gene expression. Journal of Experimental & Clinical Cancer Research, 39(1), 127.
• • Yudhani, R. D., Pesik, R. N. & Indarto, D. (2016). Metformin enhances anti-proliferative effect of cisplatin in cervical cancer cell line. Indonesian Journal of Clinical Pharmacy, 5(2), 75-83.
• • Zhang, H. H., Zhang, Y., Cheng, Y. N., Gong, F. L., Cao, Z. Q., Yu, L. G., & Guo X. L. (2018). Metformin in combination with curcumin inhibits the growth, metastasis, and angiogenesis of hepatocellular carcinoma in vitro and in vivo. Molecular Carcinogenesis, 57(1), 44-56.
• • Zhuang, Y., & Miskimins, W. K. (2011). Metformin induces both caspase- dependent and poly (ADP-ribose) polymerase-dependent cell death in breast cancer cells. Molecular Cancer Research, 9(5), 603-615.