New Beneficial Effects of Metformin on Melanoma Cancer Therapy In vivo and In vitro

New Beneficial Effects of Metformin on Melanoma Cancer Therapy In vivo and In vitro
Seyedehsarina Kalantari,^1,*
1. Farzanegan Amin Khomeini Shahr High School for Girls, Isfahan, Iran
Introduction: Melanoma is one of the most aggressive and treatment-resistant human cancer. Melanoma is the most dangerous form of skin cancer these cancerous growths develop when unrepaired DNA damage to skin cells triggers mutations in which lead the skin cells to multiply rapidly and form malignant tumors (Patel, 2011). Dacarbazine is one a not typical alkylation agent that generally considered the most active agent for remedying malignant melanoma (Crosby et al, 2000). Melanoma is known for its notorious resistance to chemotherapy, a major obstacle to successful treatment. Diabetes mellitus was first investigated as a risk factor for cancer death at the beginning of this century (El-Arabey, 2017). The association between cancer and diabetes has been a motivation for us to look for prevention strategies that can affect both diseases and reduce their overlapping load. Metformin (1,1-dimethylbiguanide hydrochloride) is used as first line therapy for diabetic mellitus type 2. The in vitro and in vivo anti-melanoma effect of metformin was investigated using B16F10 mouse melanoma cancer cell line.
Methods: Melanoma cell line B16F10 was obtained from Applied Physiology Research Center, Isfahan University of Medical Sciences. In order to achieve 80% confluence on the culture plate, 5×105 cells were plated in DMEM (10% fetal bovine serum and 1% penicillin/streptomycin) at 〖37〗^° C in a humidified atmosphere with 5% 〖CO〗_2 and rested for 48 h. The cells were incubated in 96 well flat-bottom plates (104 cell/well) for cell viability and 6 well plate (5×105 cells/well) for flow cytometric analysis. The cells were treated with dacarbazine (group 2), metformin (group 3) and combination therapy (group 4). Group 1 didn’t have any treat as a control. For in vivo experiment the mice were kept in individual cage. B16F10 were collected from cell cultures and subcutaneously implanted (3×105) in side region of mice. Seven day after incubation animals were administered metformin in drinking water (600 mg/kg) and dacarbazine intravenous injection (250 μg/kg) daily as in vitro grouping.
Results: According to our result cell viability, which was determined at various time intervals (24 and 48 h) and in the presence IC50 concentrations of the chemotherapy drug ( 0.7 mM), was reduced by ∼50% following 24 h measured in ELISA reader at 570 nm (p<0.001). Our result showed the crucial role of cancer stem cell biomarkers activation as hallmarks of cancer. High levels of CD44 were detected in all samples (around 99%) that conclude that in malignant melanoma cells, a high level expression of CD44 is associated with increased metastatic risk. This study demonstrates inhibitory effect of metformin alone and combination therapy with chemotherapeutic drug, dacarbazine, on CD44 CSCs. Also administration of metformin led to a significant reduction in tumor size compared to the group treated with dacarbazine alone. Differences between study groups were analyzed by an oneway analysis of variance (ANOVA) with a post-hoc method (SPSS 16.00).
Conclusion: These results provided that the drug combination induced significantly more apoptosis than when each drug is used individually. In addition metformin showed to target cancer stem cell and reduced CD44 expression (Breza & Magro, 2005). These data suggest metformin potential for counteracting and preventing melanoma resistance to chemotherapy. This is a very encouraging result with regard to the possibility of metformin becoming a new tool for melanoma research and treatment. According to the results of this study, metformin can be a good complementary drug to cancer chemotherapy. We believe that Metformin co-treatment with chemotherapy may prevent melanoma resistance and improve long-term survival.
Keywords: Melanoma- cancer stem cell- combination therapy