Molecular Basis of <i>Sida cordifolia</i> (L.) Induced Apoptosis in Melanoma Cell Line

Abstract

Sida cordifolia of the family Malvaceae is widely used in traditional medicine for treating inflammation, respiratory and neurological ailments and wound healing. Its extract was found to possess effective antitumor activity in hepatocellular carcinoma and HeLa cell lines. This study was aimed at screening the anticancer activity of S. cordifolia and to investigate its mechanism of action. Aerial parts of the plant were subjected to hot continuous extraction by Soxhlet apparatus with ethanol as solvent. Cytotoxicity of the extract was assessed in various cancer cell lines viz. breast, ovarian, colon, skin, and liver cancer by MTT assay. For each cell line, the IC50 value was calculated. The mechanism of anticancer activity of the extract was studied in melanoma cells by exposing them to 12.5 and 25 μg/ml extract and comparing results with the control. Gel electrophoresis was used to analyse DNA laddering. Expression of TP53, Bcl and Caspase gene family proteins were determined by SDS-PAGE. Mitochondrial membrane potential was studied by the JC-1 kit. Cell cycle analysis was performed by using a flow cytometer. Statistical analysis was done by ANNOVA, and significant values were further analysed by Tucky post-hoc analysis. P value less than 0.05 was considered statistically significant. MTT assay revealed maximum cytotoxicity of the extract against melanoma with an IC50 value of 16.51μg/ml. Melanoma cells treated with the extract demonstrated dose-dependent DNA laddering. The extract also exhibited a dose-dependent increase in the level of Bax, Caspase 3, Caspase 9 and p53 proteins. Expression of Bcl2 protein was significantly reduced. Treatment of melanoma cells with the extract showed significant loss of mitochondrial membrane potential. Melanoma cell population in subG0 and G2/S was significantly elevated. From these results, we conclude that ethanol extract of S. cordifolia is cytotoxic to melanoma cells. It acts by inducing apoptosis via an intrinsic mechanism. The extract also arrests melanoma cells in the G2/M phase.