Anticancer activity in HeLa and MCF-7 cells via apoptopic cell death by a sterol molecule Cholesta-4,6-dien-3-ol (EK-7), from the marine ascidian Eudistoma kaverium

Abstract

Objective: Eudistoma kaverium, an ascidian belonging to the family Polycitoridae is previously recorded from Indian waters. The work done on this species is limited only to taxonomy and ecology. As ascidians are potential source of anticancer compounds, we have purified fractions of E. kaverium using HPLC and evaluated its anticancer activity. Method: Chromatography based fractionation of the crude extract was carried out and mass spectrometry was used to identify the active molecule. Cell viability was checked using MTT assay. Flow cytometry, ROS generation, Hoechst staining and DNA fragmentation was performed to confirm the anticancer activity of the compound. Results: A column fraction EK-7, which was identified as a sterol Cholesta-4,6-dien-3-ol using GC–MS analysis, exhibited potent cytotoxicity with IC50 value of 6.5 μM in MCF-7 and 10.2 μM against HeLa cells. Moreover, EK-7 triggered the cell cycle arrest in the sub G0/G1 stage and induced apoptosis twenty-four hours post-treatment. Hoechst staining of EK-7-treated HeLa cells portrayed apoptotic events such as changes in cell morphology, chromatin condensation, membrane swelling, and development of apoptotic bodies. The changes in light scattering by the EK-7-treated HeLa cells indicate the general characteristics of cell death as a result of apoptosis. The EK-7-treated HeLa cells show cell cycle arrest in G0/G1, G2/M and S phases and the increased amount of sub G0/G1 population indicates the increase in the apoptotic induced cell population. The isolated compound Cholesta-4,6-dien-3-ol (EK-7), based on the GC–MS analysis and library search from E. kaverium displayed potent anticancer activity which is mediated through apoptosis targeting the G0/G1 phase. Conclusion: Based on the experimental data, it shows that EK-7(Cholesta-4,6-dien-3-ol) a sterol derivative has potent anticancer activity mediated through apoptosis. If this research is continued further with preclinical and clinical trials, EK-7(Cholesta-4,6-dien-3-ol) could be possibly used as a chemotherapeutic agent in treating cancer.

Keywords

• Eudistoma kaverium
• Ascidian
• HeLa
• MTT
• Flow cytometry
• Apoptosis