Antiproliferative effects of isoalantolactone in human liver cancer cells are mediated through caspase-dependent apoptosis, ROS generation, suppression of cell migration and invasion and targeting Ras/Raf/MEK signalling pathway

Abstract

The main objective of this study was to evaluate the in vitro antiproliferative effects of isoalantolactone against liver cancer cells (Hep-G2) and also monitor its mechanism of action. The MTT assay was involved in proliferation assessments and phase contrast microscopy was used to check cellular morphology. Acridine orange/ ethidium bromide staining along with western blotting was used to evaluate proapoptotic effects of isoalantolactone. DCFH-DA staining was used in ROS measurements. Transwell migration and invasion assay were executed to check the effects of isoalantolactone on migration and invasion of Hep-G2 cells. Western blotting was used to check the expressions of Ras/Raf/MEK signalling pathway in Hep-G2 cells. Results demonstrated that isoalantolactone significantly (*p<0.05 and **p<0.01) inhibited the proliferation of Hep-G2 cells in a concentration and time-reliant fashion. The IC50 value of the tested isoalantolactone molecule was found to be 71.2 μM and 53.4 μM at 12 h and 24 h time intervals respectively. Moreover, the antiproliferative effects of isoalantolactone were mediated through induction of caspasedependent apoptosis and oxidative stress (ROS mediated). The proapoptotic effects of isoalantolactone were evident from morphological assessments and improved expressions of caspase-3, -8, and -9 and Bax while antiapoptotic Bcl-2 was reduced significantly. Additionally, antiproliferative and proapoptotic effects of isoalantolactone were found to be a consequence of blocking of Ras/Raf/MEK signalling in Hep-G2 cells. Furthermore, isoalantolactone significantly (*p<0.05) targeted the migration and invasion of Hep-G2 cells. In conclusion, these results validated that isoalantolactone shows strong antiproliferative activity against Hep-G2 liver cancer cells. Therefore, it could prove as a leading candidate in liver cancer research, drug discovery and design.