Metformin Modulates Cyclin D1 and P53 Expression to Inhibit Cell Proliferation and to Induce Apoptosis in Cervical Cancer Cell Lines

Abstract

Background: Cervical cancer is one of the most prevalent gynecological cancers worldwide and contributes in high mortality of Indonesian women. The efficacy of chemotherapy as a standart therapy for cervical cancer decreases because it frequenly rises adverse effects. Recent studies have found that metformin has a potential anticancer effect mostly through reduction of cyclin expression and activation of Activated Adenosine Monophosphate Kinase (AMPK). This study aimed to investigate the effect of metfomin on expression of cyclin D1 and p53 and apoptosis in HeLa cancer cell line. Methods: HeLa cells were treated with various doses of metformin and doxorubicin as a positive control. Cytotoxic effect of metformin was determined using the MTT assay. Immunocytochemistry was used to assess cyclin D1 and p53 expression and apoptosis levels of treated HeLa cells were analyzed using flowcytometry. Data of cyclin D1 expression was statistically analyzed using the Kruskal-Wallis test followed by the Tamhane test, whilst ANOVA and Tukey post Hoc tests were used to analyze data of p53 and apoptosis level. The significant value was p< 0.05. Results: Metformin was able to inhibit proliferation of HeLa cells with IC50 60 mM. HeLa cells treated with 60 and 120 mM metformin had lower cyclin D1 expression than HeLa cells treated without metformin and reached a significant difference (p= 0.001). Moreover, 30 mM or higher doses of metformin increase significantly p53 expression (p< 0.001). Induction of apoptosis was observed in HeLa cells treated with all doses of metformin and reached statistically difference (p= 0.04 and p < 0.001). Conclusion: Metformin can modulate cyclin D1 and p53 expression in HeLa cancer cell line, leading to inhibition of cell proliferation and induction of apoptosis. Other cyclin family members, CDK inhibitors and AMPK signaling should be further investigated in order to know mechanism of metformin action.

Keywords: Apoptosis; Cyclin D1; HeLa cell; Metformin; p53.

Figure 1

Cyclin D1 Expression in HeLa Cells Treated with Metformin. 2 x 10⁵ HeLa cells were grown on to a coverslip treated with (a) 0 mM, (b) 30 mM, (c) 60 mM, and (d) 120 mM metformin. Cells expressing cyclin D1 were analyzed by using immunocytochemistry with antibody anti human cyclin D1. Brown-stained cells (red arrows) were calculated from 5 point views with x400 magnification and all data were presented as percentage ± standard deviation (e). Experiments were performed in triplicate. *Significant value between treatmen and control group were set up at p < 0.05

Figure 2

p53 Expression in HeLa Cells Treated with Metformin. 2 x 10⁵ HeLa cells were grown on to a coverslip treated with (a) 0 mM, (b) 30 mM, (c) 60 mM, and (d) 120 mM metformin. Cells expressing p53 were analyzed by using immunocytochemistry with antibody anti human p53. Brown-stained cells (red arrows) were calculated from 5 point views with x400 magnification and all data were presented as percentage ± standard deviation (e). Experiments were performed in triplicate. *Significant value between treatment and control group were set up at p < 0.05

Figure 3

Apoptosis in HeLa cells treated with metformin. 2 x 10⁵ HeLa cells were incubated for 48 h in the presence of 30 mM (b), 60 mM (c) and 120 mM (d) metformin whilst cell only was a negative control (a) and 15 µM doxorubicin (e) was positive control. Apoptosis levels were analyzed using flow cytometry. Results represent three independent experiments (f). *Significant value between treatment and control group were set up at p < 0.05