Synergistic Action of 1,2-Epoxy-3 (3- (3,4-dimethoxyphenyl)- 4H-1-benzopiyran-4-on) Propane with Doxorubicin and Cisplatin through Increasing of p53, TIMP-3, and MicroRNA-34a in Cervical Cancer Cell Line (HeLa)

Abstract

Objective:Cervical cancer is the second most common cancer among women worldwide, with a high mortality rate especially in developing countries. Insufficient treatment for cervical cancer, multiple side effects, and high drug prices encourage researchers to look for effective and selective cancer drugs with appropriate molecular targets. This study explored the cytotoxicity of (1,2-epoxy-3(3-(3,4-dimethoxyphenyl)-4H-1-benzopyran-4-on) propane (EPI) synthesized from clove leaves oil on HeLa cells, its combination with doxorubicin (DOX) and cisplatin (CIS), and also their influence on p53, TIMP-3, and miR-34a as therapeutic targets.Materials and Methods:This research was an experimental in vitro study on cervical cancer uteri culture. The cytotoxicity was analyzed by MTT assay. The drug combination synergisms were indicated by the combination index (CI) (using CompuSyn 1.4). HeLa cells in 32 wells were divided into eight groups as negative control, which were given EPI ½IC50, EPI IC50, EPI 2IC50, DOX IC50, combination of EPI+DOX, CIS, and the combination of EPI+CIS. The p53 and TIMP-3 concentrations were measured using ELISA, and expressions of miR-34a with qRT-PCR. One-way ANOVA and post hoc Tukey tests were performed to determine the mean difference of all variables between the study groups.Results:IC50 for EPI was 33.24 (±3.01) μg/ml, while DOX and CIS were 4.8 μg/ml (±0.1), and 23.34 μg/ml (±3.01), respectively, while CI values for EPI-DOX were <0.1 and for EPI-CIS <0.9. Expression of p53 in group 6 (1.67±0.31) μg/ml and 8 (1.18±0.18) μg/ml, TIMP-3 6 (3.81±0.49) µg/ml and 8 (2.93±0.42) µg/ml were significantly higher compared to the control group (p<0.05). All treatment groups showed significantly increased miR-34a expressions compared to the control group (p<0.05).Conclusion:The combinations showed a very strong synergism and a moderate slight synergism for EPI-DOX and EPI-CIS. Both combinations were able to increase the expressions of p53, TIMP-3 proteins, and MiR-34a in the HeLa cells.