Kazım ŞENOL, Güven YENMİŞ, Fatma Sinem HOCAOĞLU EMRE, Elif YAPRAK SARAÇ, Nail Beşli, Gönül KANIGÜR SULTUYBEK

Metformin promotes apoptosis in primary breast cancer cells by downregulation of cyclin D1 and upregulation of P53 through an AMPK-alpha independent mechanism

Background/aim: In the present study we aimed to figure out the effect of metformin on the expression of AMPK-alpha, cyclin D1, and Tp53, and apoptosis in primary breast cancer cells (PBCCs). Materials and methods: PBCCs were treated with two doses of metformin (0 mM, 25 mM). Proliferation was determined by BrdU assay. Real-time PCR was used to assess AMPK-alpha, cyclin D1, and Tp53 gene expressions; apoptotic indexes of PBCCs were analyzed using flow-cytometry. Results: Twenty-four–hour incubation with 25 mM metformin reduced the proliferation of PBCCs. AMPK-alpha gene expression in PBCCs was not affected by 25 mM metformin treatment compared with the control group. PBCCs treated with 25 mM metformin had lower cyclin D1 expression compared with nontreated cells; however, the difference was not statistically significant. Twenty-five millimolar dose of metformin increased p53 expression significantly compared with the nontreated group. The high concentration of metformin elevated the number of annexin V-positive apoptotic cells, and the increase in the apoptotic index was statistically significant. Conclusion: Metformin can modulate cyclin D1 and p53 expression through AMPK-alpha-independent mechanism in breast cancer cells, leading to cell proliferation inhibition and apoptosis induction.Key words: AMPK-alpha, cyclin D1, Tp53, breast cancer, metformin

PDF

___

1. Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS et al. Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. The New England Journal of Medicine 1998; 338: 867-872. doi: 10.1056/NEJM199803263381303
2. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P et al. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 2002; 51: 2074-2081. doi: 10.2337/diabetes.51.7.2074
3. Stumvoll M, Nurjhan N, Perriello G, Dailey G, Gerich JE. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. The New England Journal of Medicine 1995; 333: 550-554. doi: 10.1056/NEJM199508313330903
4. Ouyang J, Parakhia RA, Ochs RS. Metformin activates amp kinase through inhibition of amp deaminase. Journal of Biological Chemistry 2011; 286: 1-11.. doi: 10.1074/jbc.M110.121806
5. Zhou G, Myers R, Li Y, Chen Y, Shen X et al. Role of amp-activated protein kinase in mechanism of metformin action. Journal of Clinical Investigation 2001; 108: 1167-1174. doi: 10.1172/JCI13505
6. Wang Y, Lin B, Wu J, Zhang H, Wu B. Metformin inhibits the proliferation of A549/CDDP cells by activating p38 mitogenactivated protein kinase. Oncology Letters 2014; 8: 1269-1274. doi: 10.3892/ol.2014.2270
7. Ashinuma H, Takiguchi Y, Kitazono S, Kitazono-Saitoh M, Kitamura A et al. Antiproliferative action of metformin in human lung cancer cell lines. Oncology Reports 2012; 28: 8-14. doi: 10.3892/ or.2012.1763
8. Storozhuk Y, Hopmans SN, Sanli T, Barron C, Tsiani E et al. Metformin inhibits growth and enhances radiation response of nonsmall cell lung cancer (NSCLC) through ATM and AMPK. British Journal of Cancer 2013; 108: 2021-2032. doi: 10.1038/bjc.2013.187
9. Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P et al. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene 2008; 27: 3576-3586. doi: 10.1038/sj.onc.1211024
10. Alimova IN, Liu B, Fan Z, Edgerton SM, Dillon T et al. Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 2009; 8: 909-915. doi: 10.4161/ cc.8.6.7933
11. Zhang T, Guo P, Zhang Y, Xiong H, Yu X et al. The antidiabetic drug metformin inhibits the proliferation of bladder cancer cells in vitro and in vivo. International Journal of Molecular Sciences 2013; 14: 24603-24618. doi: 10.3390/ijms141224603
12. Al-Maghrabi J, Al-Sakkaf K, Qureshi IA, Butt NS, Damnhory L et al. AMPK expression patterns are significantly associated with poor prognosis in breast cancer patients. Annals of Diagnostic Pathology 2017 Aug; 29: 62-67. doi: 10.1016/j.anndiagpath.2017.05.012. Epub 2017 May 16.
13. Hadad SM, Hardie DG, Appleyard V, Thompson AM. Effects of metformin on breast cancer cell proliferation, the AMPK pathway and the cell cycle. Clinical and Translational Oncology 2014 Aug;16(8):746-752. doi: 10.1007/s12094-013-1144-8. Epub 2013 Dec 12.
14. Ben Sahra I, Laurent K, Giuliano S, Larbret F, Ponzio G et al. Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells. Cancer Research 2010; 70: 2465-2475.. doi: 10.1158/0008-5472. CAN-09-2782
15. Zhuang Y, Miskimins WK. Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1. Journal of Molecular Signaling 2008; 3: 18. doi: 10.1186/1750-2187-3-18
16. Malki A, Youssef A. Antidiabetic drug metformin induces apoptosis in human MCF breast cancer via targeting ERK signaling. Oncology Research 2011; 19: 275-85.. doi: 10.3727/096504011x130 21877989838
17. Janjetovic K, Harhaji-Trajkovic L, Misirkic-Marjanovic M, Vucicevic L, Stevanovic D et al. In vitro and in vivo anti-melanoma action of metformin. European Journal of Pharmacology 2011; 668: 373-382. doi: 10.1016/j.ejphar.2011.07.004
18. Nelson LE, Valentine RJ, Cacicedo JM, Gauthier MS, Ido Y et al. A novel inverse relationship between metformin-triggered AMPKSIRT1 signaling and p53 protein abundance in high glucose-exposed HepG2 cells. American Journal of Physiology-Cell Physiology 2012; 303: C4-13.. doi: 10.1152/ajpcell.00296.2011
19. Cerezo M, Tichet M, Abbe P, Ohanna M, Lehraiki A et al. Metformin blocks melanoma invasion and metastasis development in AMPK/p53-dependent manner. Molecular Cancer Therapeutics 2013;12:1605-15. doi: 10.1158/1535-7163.MCT-12-1226-T
20. Yi G, He Z, Zhou X, Xian L, Yuan T et al. Low concentration of metformin induces a p53-dependent senescence in hepatoma cells via activation of the AMPK pathway. International Journal of Oncology 2013; 43: 1503-1510. doi: 10.3892/ijo.2013.2077
21. Gerl R and Vaux DL. Apoptosis in the development and treatment of cancer. Carcinogenesis 2005; 26(2): 263-270. doi: 10.1093/carcin/ bgh283
22. Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis pathways in cancer therapy. CA: A Cancer Journal for Clinicians 2005; 55: 178- 194. doi: 10.3322/canjclin.55.3.178
23. Malecki M, Sabo C, Foorohar A, Tombokan X. Novel paradigm for immunotherapy of breast cancer by engaging prophylactic immunity against hepatitis B. Clinical and Translational Medicine 2016; 5(1): 32. doi: 10.1186/s40169-016-0111-8
24. Varghese S, Samuel SM, Varghese E, Kubatka P, Büsselberg D. High glucose represses the anti-proliferative and pro-apoptotic effect of metformin in triple negative breast cancer cells. Biomolecules 2019; 9(1): 16. doi: 10.3390/biom9010016
25. Sena P, Mancini S, Benincasa M, Mariani F, Palumbo C, Roncucci L. Metformin induces apoptosis and alters cellular responses to oxidative stress in Ht29 colon cancer cells: preliminary findings. International Journal of Molecular Sciences 2018; 19(5): 1478. doi: 10.3390/ijms19051478
26. Liu B, Fan Z, Edgerton SM, Deng XS, Alimova IN et al. Metformin induces unique biological and molecular responses in triple negative breast cancer cells. Cell Cycle 2009; 8: 2031-2040. doi: 10.4161/ cc.8.13.8814
27. Kisfalvi K, Eibl G, Sinnett-Smith J, Rozengurt E. Metformin disrupts crosstalk between G protein-coupled receptor and insulin receptor signaling systems and inhibits pancreatic cancer growth. Cancer Research 2009; 69: 6539-6545. doi: 10.1158/0008-5472.CAN09-0418
28. Goodwin PJ, Pritchard KI, Ennis M, Clemons M, Graham M et al. Insulin-lowering effects of metformin in women with early breast cancer. Clinical Breast Cancer 2008; 8: 501-505. doi: 10.3816/ CBC.2008.n.060
29. Isakovic A, Harhaji L, Stevanovic D, Markovic Z, SumaracDumanovic M et al. Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis. Cellular and Molecular Life Sciences 2007; 64: 1290-1302. doi: 10.1007/s00018- 007-7080-4
30. Saito S, Furuno A, Sakurai J, Sakamoto A, Park HR et al. Chemical genomics identifies the unfolded protein response as a target for selective cancer cell killing during glucose deprivation. Cancer Research 2009; 69: 4225-4234. doi: 10.1158/0008-5472.CAN08-2689
31. Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G et al. Metformininduced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochemical Pharmacology 2004; 68: 409-416. doi: 10.1016/j. bcp.2004.04.003
32. Yang YC, Chien MH, Liu HY, Chang YC, Chen CK et al. Nuclear translocation of PKM2/AMPK complex sustains cancer stem cell populations under glucose restriction stress. Cancer letters 2018; 421: 28-40. doi: 10.1016/j.canlet.2018.01.075
33. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes & Development 1993; 7: 812-821. doi: 10.1101/gad.7.5.812
34. Vermeulen K, Berneman ZN, Bockstaele DRVn. Cell cycle and apoptosis. Cell Proliferation 2003; 36: 165-175. doi: 10.1046/j.1365- 2184.2003.00267.x
35. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J et al. AMP-activated protein kinase induces a p53-Dependent metabolic checkpoint. Molecular Cell 2005; 18: 283-293.. doi: 10.1016/j.molcel.2005.03.027
36. Queiroz EA, Puukila S, Eichler R, Sampaio SC, Forsyth HL et al. Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells. PLoS One 2014; 9(5): e98207. doi: 10.1371/journal.pone.0098207
37. Wang LW, Li ZS, Zou DW, Jin ZD, Gao J, Xu GM. Metformin induces apoptosis of pancreatic cancer cells. World Journal of Gastroenterology 2008; 14: 7192-7198. doi: 10.3748/wjg.14.7192
38. Rattan R, Giri S, Hartmann LC, Shridhar V. Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner. Journal of Cellular and Molecular Medicine 2011; 15: 166-178.. doi: 10.1111/j.1582-4934.2009.00954.x
39. Kato K, Gong J, Iwama H, Kitanaka A, Tani J et al. The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo. Molecular Cancer Therapeutics 2012; 11: 549-560. doi: 10.1158/1535-7163.MCT-11-0594
40. Ben Sahra I, Regazzetti C, Robert G, Laurent K, Le MarchandBrustel Y et al. Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Cancer Research 2011; 71: 4366-4372. doi: 10.1158/0008-5472.CAN-10-1769
41. Zhuang Y, Chan DK, Haugrud AB, Miskimins WK. Mechanisms by which low glucose enhances the cytotoxicity of metformin to cancer cells both in vitro and in vivo. PloS One 2014; 9:e108444. doi: 10.1371/journal.pone.0108444