Sains Malaysiana 50(5)(2021): 1393-1405

http://doi.org/10.17576/jsm-2021-5005-18

Anti-Tumor Activity of Metformin in Human Epidermal Growth Factor Receptor 2 Positive Breast Cancer Cells

(Aktiviti Anti-Tumor Metformin pada Reseptor Faktor Pertumbuhan Epidermis Manusia 2 Positif Sel Kanser Payudara)

FATHUL HUDA^1,2*, SARI EKAWATI³, ANINDY PUTRI ADDINA³, AHMAD FARIED^2,4, AFIAT BERBUDI^1,2, TAOFIK RUSDIANA⁵, TENNY PUTRI⁶, NURUL QOMARILLA⁶, LUKMAN HILFI⁷, IWAN SETIAWAN¹ & MUHAMMAD HASAN BASHARI^1,2

¹Department of Biomedical Science, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

²Oncology and Stem Cell Working Group, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

³Bachelor of Medicine Program, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

⁴Department of Neurosurgery, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

⁵Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Universitas Padjadjaran, Bandung 40161, Indonesia

⁶Cell Culture Laboratory, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

⁷Department of Public Health, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

Diserahkan: 18 Disember 2019/Diterima: 29 September 2020

ABSTRACT

Breast Cancer (BC) is the leading cause of cancer death in women. One BC subtype is very aggressive with amplification of human epidermal growth factor receptor 2 (HER2) protein. Although specific HER2+ targeting agents are available, most of HER2+ BC patients develop resistant to these agents. Recent studies show that metformin, is able to become anti-tumor in various cancer cells. This research aims to evaluate anti-tumor activities of metformin to HER2+ BC cells in both sensitive and resistant to trastuzumab. A series of assays were performed to evaluate metformin anti-tumor activities in HCC-1954 and SKBR-3 HER2+ BC cells. MTT assay was performed to evaluate cell death, and inhibitory concentration (IC₅₀), while scratch assay was performed to assess inhibition of cell migration and clonogenic assay to assess cell proliferation. p<0.05 was considered to be significant. Metformin could suppress the number of HER2+ BC cells. Viability assay showed suppression of viable cells after metformin incubation of 60 and 600 µM compared to control, 30 and 90%, respectively. Surprisingly, IC₅₀ of metformin was smaller in HER2+ BC HCC-1954 cells that resistant to trastuzumab compare to the sensitive one (SKBR-3). Both were below 1 µM, with R² more than 0.95. Additionally, clonogenic assay showed less colony number and colony area with at least p < 0.05 in colony number and p < 0.01 in the area. In addition, metformin inhibited cell migration of HER2+ BC cells. Metformin shows a potency as anti-tumor by inducing cell death, inhibiting cell proliferation and cell migration of HER2+ BC cells.

Keywords: Breast Cancer; HER2+; metformin; Trastuzumab resistant

ABSTRAK

Kanser Payudara (BC) adalah penyebab utama kematian akibat barah pada wanita. Satu subjenis BC sangat agresif dengan penguatan protein reseptor 2 faktor pertumbuhan epidermis manusia (HER2). Walaupun agen sasaran HER2 + khusus ada, kebanyakan pesakit HER2 + BC mengalami ketahanan terhadap agen ini. Kajian terbaru menunjukkan bahawa metformin mampu menjadi anti-tumor pada pelbagai sel barah. Penyelidikan ini bertujuan untuk menilai aktiviti anti-tumor metformin kepada sel HER2 + BC dengan sensitif dan tahan terhadap trastuzumab. Satu siri ujian dilakukan untuk menilai aktiviti anti-tumor metformin pada sel HCC-1954 dan SKBR-3 HER2 + BC. Ujian MTT dilakukan untuk menilai kematian sel dan kepekatan penghambatan (IC₅₀), sementara ujian awal dilakukan untuk menilai penghambatan penghijrahan sel dan pengujian klonogenik untuk menilai percambahan sel. p <0.05 dianggap signifikan. Metformin dapat menekan bilangan sel HER2 + BC. Ujian daya maju menunjukkan penekanan sel yang berdaya maju setelah inkubasi metformin 60 dan 600 µM berbanding kawalan, masing-masing 30 dan 90%. Anehnya, IC50 metformin lebih kecil pada sel HER2 + BC HCC-1954 yang tahan terhadap trastuzumab dibandingkan dengan sel sensitif (SKBR-3). Kedua-duanya berada di bawah 1 µM, dengan R2 lebih daripada 0.95. Selain itu, ujian klonogenik menunjukkan bilangan koloni dan kawasan koloni yang kurang dengan sekurang-kurangnya p <0.05 pada bilangan koloni dan p < 0.01 di kawasan koloni. Sebagai tambahan, metformin menghalang penghijrahan sel HER2 + BC sel. Metformin menunjukkan potensi sebagai anti-tumor dengan mendorong kematian sel, menghambat percambahan sel dan penghijrahan sel HER2+ sel BC.

Kata kunci: HER2 +; kanser payudara (BC); metformin, rintangan Trastuzumab

RUJUKAN

Bashari, M.H., Fan, F., Vallet, S., Sattler, M., Arn, M., Luckner-Minden, C., Schulze-Bergkamen, H., Zornig, I., Marme, F., Schneeweiss, A., Cardone, M.H., Opferman, J.T., Jager, D. & Podar, K. 2016. Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: Therapeutic implications. Breast Cancer Research 18(1): 26.

Bowker, S.L., Majumdar, S.R., Veugelers, P. & Johnson, J.A. 2006. Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin. Diabetes Care 29(2): 254-258.

Buzzai, M., Jones, R.G., Amaravadi, R.K., Lum, J.L., DeBerardinis, R.J., Zhao, F., Viollet, B. & Thompson, C.B. 2007. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Research 67(14): 6745-6752.

Cazzaniga, M., DeCensi, A., Pruneri, G., Puntoni, M., Bottiglieri, L., Varricchio, C., Guerrieri-Gonzaga, A., Gentilini, O.D., Pagani, G. & Dell'Orto, P. 2013. The effect of metformin on apoptosis in a breast cancer presurgical trial. British Journal of Cancer 109(11): 2792-2797.

Cazzaniga, M., Bonanni, B., Guerrieri-Gonzaga, A. & Decensi, A. 2009. Is it time to test metformin in breast cancer clinical trials? Cancer Epidemiology and Prevention Biomarkers 18(3): 701-705.

Chung, Y.C., Chang, C.M., Wei, W.C., Chang, T.W., Chang, K.J. & Chao, W.T. 2018. Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells. Scientific Report 8(1): 3930.

Cobleigh, M.A., Vogel, C.L., Tripathy, D., Robert, N.J., Scholl, S., Fehrenbacher, L., Wolter, J.M., Paton, V., Shak, S. & Lieberman, G. 1999. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Journal of Clinical Oncology 17(9): 2639.

Collier, C.A., Bruce, C.R., Smith, A.C., Lopaschuk, G. & Dyck, D.J. 2006. Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle. American Journal of Physiology-Endocrinology and Metabolism 291(1): E182-E189.

Davies, G., Lobanova, L., Dawicki, W., Groot, G., Gordon, J.R., Bowen, M., Harkness, T. & Arnason, T. 2017. Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer. PLoS ONE 12(12): e0187191.

Della Corte, C.M., Ciaramella, V., Di Mauro, C., Castellone, M.D., Papaccio, F., Fasano, M., Sasso, F.C., Martinelli, E., Troiani, T., De Vita, F., Orditura, M., Bianco, R., Ciardiello, F. & Morgillo, F. 2016. Metformin increases antitumor activity of MEK inhibitors through GLI1 downregulation in LKB1 positive human NSCLC cancer cells. Oncotarget 7(4): 4265-4278.

Deng, J., Peng, M., Wang, Z., Zhou, S., Xiao, D., Deng, J., Yang, X., Peng, J. & Yang, X. 2019. Novel application of metformin combined with targeted drugs on anticancer treatment. Cancer Science 110(1): 23-30.

Diaz-Rodriguez, E., Perez-Pena, J., Rios-Luci, C., Arribas, J., Ocana, A. & Pandiella, A. 2019. TRAIL receptor activation overcomes resistance to trastuzumab in HER2 positive breast cancer cells. Cancer Letters 453: 34-44.

Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D. & Bray, F. 2015. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer 136(5): E359-E386.

Franken, N.A.P., Rodermond, H.M., Stap, J., Haveman, J. & Bree, C.V. 2006. Clonogenic assay of cells in vitro. Nature Protocols 1(5): 2315-2319.

Gallagher, E.J. & LeRoith, D. 2011. Diabetes, cancer, and metformin: Connections of metabolism and cell proliferation. Annals of the New York Academy of Sciences 1243(1): 54-68.

Gao, Z.Y., Liu, Z., Bi, M.H., Zhang, J.J., Han, Z.Q., Han, X., Wang, H.Y., Sun, G.P. & Liu, H. 2016. Metformin induces apoptosis via a mitochondria-mediated pathway in human breast cancer cells in vitro. Experimental and Therapeutic Medicine 11(5): 1700-1706.

Guo, Z., Sevrioukova, I.F., Denisov, I.G.,  Zhang, X., Chiu, T.L., Thomas, D.G., Hanse, E.A., Cuellar, R.A.D., Grinkova, Y.G., Langenfeld, V.W., Swedien, D.S., Stamschror, J.D., Alvarez, J., Luna, F., Galvan, A., Bae, Y.K., Wulfkuhle, J.D., Gallagher, R.I., Rd Petricoin, E.F., Norris, B., Flory, C.M., Schumacher, R.J., O'Sullivan, M.G., Cao, Q., Chu, H., Lipscomb, J.D., Atkins, W.M., Gupta, K., Kelekar, A., Blair, I.A., Capdevila, J.H., Falck, J.R., Sligar, S.G., Poulos, T.L., Georg, G.I., Ambrose, E. & Potter, D.A. 2017. Heme binding biguanides target cytochrome P450-dependent cancer cell mitochondria. Cell Chemical Biology 24(10): 1259-1275.

Guzmán, C., Bagga, M., Kaur, A., Westermarck, J. & Abankwa, D. 2014. ColonyArea: An ImageJ plugin to automatically quantify colony formation in clonogenic assays. PLoS ONE 9(3): e92444.

Hacioglu, B., Akin, S., Sever, A.R. & Altundag, K. 2015. Clinical implications of intratumoral heterogeneity of HER2 gene amplification in locally advanced HER2-positive breast cancer patients. Future Oncology 11(18): 2495-2497.

Hadad, S.M., Hardie, D.G., Appleyard, V. & Thompson, A.M. 2014. Effects of metformin on breast cancer cell proliferation, the AMPK pathway and the cell cycle. Clinical and Translational Oncology 16(8): 746-752.

Holliday, D.L. & Speirs, V. 2011. Choosing the right cell line for breast cancer research.  Breast Cancer Research 13(4): 215.

Iqbal, N. & Iqbal, N. 2014. Human epidermal growth factor receptor 2 (HER2) in cancers: Overexpression and therapeutic implications. Molecular Biology International 2014: Article ID. 852748.

Jang, S.Y., Kim, A., Kim, J.K., Kim, C., Cho, Y.H., Kim, J.H., Kim, C.H. & Lee, J.Y. 2014. Metformin inhibits tumor cell migration via down-regulation of MMP9 in tamoxifen-resistant breast cancer cells. Anticancer Research 34(8): 4127-4134.

Jiralerspong, S., Palla, S.L., Giordano, S.H., Meric-Bernstam, F., Liedtke, C., Barnett, C.M., Hsu, L., Hung, M.C., Hortobagyi, G.N. & Gonzalez-Angulo, A.M. 2009. Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. Journal of Clinical Oncology 27(20): 3297-3302.

Kasznicki, J., Sliwinska, A. & Drzewoski, J. 2014. Metformin in cancer prevention and therapy. Annals of Translational Medicine 2(6): 57.

Lavaud, P. & Andre, F. 2014. Strategies to overcome trastuzumab resistance in HER2-overexpressing breast cancers: Focus on new data from clinical trials. BMC Medicine 12: 132.

Li, W.D., Li, N.P., Song, D.D., Rong, J.J., Qian, A.M. & Li, X.Q. 2017. Metformin inhibits endothelial progenitor cell migration by decreasing matrix metalloproteinases, MMP-2 and MMP-9, via the AMPK/mTOR/autophagy pathway. International Journal of Molecular Medicine 39(5): 1262-1268.

Liu, B., Fan, Z., Edgerton, S.M., Yang, X., Lind, S.E. & Thor, A.D. 2011. Potent anti-proliferative effects of metformin on trastuzumab-resistant breast cancer cells via inhibition of erbB2/IGF-1 receptor interactions. Cell Cycle 10(17): 2959-2966.

Lord, S.R., Cheng, W.C., Liu, D., Gaude, E., Haider, S., Metcalf, T., Patel, N., Teoh, E.J., Gleeson, F., Bradley, K., Wigfield, S., Zois, C., McGowan, D.R., Ah-See, M.L., Thompson, A.M., Sharma, A., Bidaut, L., Pollak, M., Roy, P.G., Karpe, F., James, T., English, R., Adams, R.F., Campo, L., Ayers, L., Snell, C., Roxanis, I., Frezza, C., Fenwick, J.D., Buffa, F.M. & Harris, A.L. 2018. Integrated pharmacodynamic analysis identifies two metabolic adaption pathways to metformin in breast cancer. Cell Metabolism 28(5): 679-688.e4.

Malek, M., Aghili, R., Emami, Z. & Khamseh, M.E. 2013. Risk of cancer in diabetes: The effect of metformin. ISRN Endocrinology 2013: Article ID. 636927.

Marcotte, R., Sayad, A., Brown, K.R., Sanchez-Garcia, F., Reimand, J., Haider, M., Virtanen, C., Bradner, J.E., Bader, G.D. & Mills, G.D. 2016. Functional genomic landscape of human breast cancer drivers, vulnerabilities, and resistance. Cell 164(1): 293-309.

Marinello, P.C., Panis, C., Silva, T.N.X., Binato, R., Abdelhay, E., Rodrigues, J.A., Mencalha, A.L., Lopes, N.M.D., Luiz, R.C., Cecchini, R. & Cecchini, A.L. 2019. Metformin prevention of doxorubicin resistance in MCF-7 and MDA-MB-231 involves oxidative stress generation and modulation of cell adaptation genes.  Scientific Report 9(1): 5864.

Martin-Castillo, B., Dorca, J., Vazquez-Martin, A., Oliveras-Ferraros, C., Lopez-Bonet, E., Garcia, M., Barco, S.D. & Menendez, J.A. 2010. Incorporating the antidiabetic drug metformin in HER2-positive breast cancer treated with neo-adjuvant chemotherapy and trastuzumab: An ongoing clinical-translational research experience at the Catalan Institute of Oncology. Annals of Oncology 21(1): 187-189.

Parker, J.S., Mullins, M., Cheang, M.C.U., Leung, S., Voduc, D., Vickery, S., Davies, S., Fauron, C., He, X. & Hu, Z. 2009. Supervised risk predictor of breast cancer based on intrinsic subtypes. Journal of Clinical Oncology 27(8): 1160-1167.

Pernicova, I. & Korbonits, M. 2014. Metformin--mode of action and clinical implications for diabetes and cancer. Nature Reviews Endocrinology 10(3): 143-156.

Pierro, J., Saba, C., McLean, K., Williams, R., Karpuzoglu, E., Prater, R., Hoover, K. & Gogal, R. 2017. Anti-proliferative effect of metformin on a feline injection site sarcoma cell line independent of Mtor inhibition. Research in Veterinary Science 114: 74-79.

Quattrini, I., Conti, A., Pazzaglia, L., Novello, C., Ferrari, S., Picci, P. & Benassi, M.S. 2014. Metformin inhibits growth and sensitizes osteosarcoma cell lines to cisplatin through cell cycle modulation. Oncology Reports 31(1): 370-375.

Rezano, A., Kuwahara, K., Yamamoto-Ibusuki, M., Kitabatake, M., Moolthiya, P., Phimsen, S., Suda, T., Tone, S., Yamamoto, Y., Iwase, H. & Sakaguchi, N. 2013. Breast cancers with high DSS1 expression that potentially maintains BRCA2 stability have poor prognosis in the relapse-free survival. BMC Cancer 13: 562.

Scherbakov, A.M., Sorokin, D.V., Tatarskiy Jr., V.V., Prokhorov, N.S., Semina, S.E., Berstein, L.M. & Krasil'nikov, M.A. 2016. The phenomenon of acquired resistance to metformin in breast cancer cells: The interaction of growth pathways and estrogen receptor signaling. IUBMB Life 68(4): 281-292.

Sharma, A., Bandyopadhayaya, S., Chowdhury, K., Sharma, T., Maheshwari, R., Das, A., Chakrabarti, G., Kumar, V. & Mandal, C.C. 2019. Metformin exhibited anticancer activity by lowering cellular cholesterol content in breast cancer cells. PLoS ONE 14(1): e0209435.

Sharma, G.N., Dave, R., Sanadya, J., Sharma, P. & Sharma, K.K. 2010. Various types and management of breast cancer: An overview. Journal of Advanced Pharmaceutical and Technological Research 1(2): 109-126.

Silvestri, A., Palumbo, F., Rasi, I., Posca, D., Pavlidou, T., Paoluzi, S., Castagnoli, L. & Cesareni, G. 2015. Metformin induces apoptosis and downregulates pyruvate kinase M2 in breast cancer cells only when grown in nutrient-poor conditions. PLoS ONE 10(8): e0136250.

Strober, W. 2015. Trypan blue exclusion test of cell viability. Current Protocols in Immunology 111: A3.B.1-A3.B.3.

Subik, K., Lee, J.F., Baxter, L., Strzepek, T., Costello, D., Crowley, P., Xing, L., Hung, M.C., Bonfiglio, T., Hicks, D.G. & Tang, P. 2010. The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer 4: 35-41.

Vazquez-Martin, A., Oliveras-Ferraros, C., Del Barco, S., Martin-Castillo, B. & Menendez, J.A. 2011. The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells. Breast Cancer Research Treatment 126(2): 355-364.

Viollet, B., Guigas, B., Garcia, N.S., Leclerc, J., Foretz, M. & Andreelli, F. 2012. Cellular and molecular mechanisms of metformin: An overview. Clinical science 122(6): 253-270.

Wahidin, M., Noviani, R., Hermawan, S., Andriani, V., Ardian, A. & Djarir, H. 2012. Population-based cancer registration in Indonesia. Asian Pacific Journal of Cancer Prevention 13(4): 1709-1710.

Wang, L.W., Li, Z.S., Zou, D.W., Jin, Z.D., Gao, J. & Xu, G.M. 2008. Metformin induces apoptosis of pancreatic cancer cells. World Journal of Gastroenterology 14(47): 7192-7198.

Zhu, P., Davis, M., Blackwelder, A.J., Bachman, N., Liu, B., Edgerton, S., Williams, L.L., Thor, A.D. & Yang, X. 2014. Metformin selectively targets tumor-initiating cells in ErbB2-overexpressing breast cancer models. Cancer Prevention Research 7(2): 199-210.

*Pengarang untuk surat-menyurat; email: fathul@unpad.ac.id