Tormentic acid induces anticancer effects in cisplatin-resistant human cervical cancer cells mediated via cell cycle arrest, ROS production, and targeting mTOR/PI3K/AKT signalling pathway

Purpose: Tormentic acid has been shown to exert remarkable anti-cancer potential against different cancer cell types. In this study, the anti-cancer potential of tormentic acid was examined in cisplatin-resistant cervical cancer cells (HeLa cells). Further, the effects of tormentic acid on cell cycle, reactive oxygen species (ROS) production, and mTOR/PI3K/AKT signalling pathway were evaluated as well. Methods: Cell viability was evaluated by MTT assay and its impact on mTOR/PI3K/AKT signalling pathway was estimated via western blot assay. Colony formation was analysed through clonogenic assay and phase-contrast microscopy was used for the determination of apoptotic cell morphology along with DAPI staining. Fluorescence-activated cell sorting was performed for cell cycle analysis and ROS production was monitored by fluorescence microscopy. Results: The results indicated that tormentic acid significantly suppresses the proliferation of HeLa cells. These antiproliferative effects of tormentic acid were dose-dependent. Clonogenic assay revealed anti-colony formation potential of tormentic acid. Tormentic acid also induced remarkable morphological changes in HeLa cells, indicative of apoptosis. Further, DAPI staining assay showed formation of apoptotic bodies along with dead cells bearing apoptotic nuclei. Western blotting showed impressive increase in the expressions of pro-apoptotic proteins and decreased expression of anti-apoptotic proteins. Fluorescence-activated cell sorting (FACS) analysis revealed that tormentic acid induced G2/M phase cell cycle arrest and its effectiveness increased with increased doses. Fluorescence intensity indicated amplified ROS production after tormentic acid exposure. The expression of tormentic acid on mTOR/PI3K/AKT pathway revealed blocking of this pathway with a concentration-dependent manner. Conclusions: The outcomes of the present investigation suggest that tormentic acid-induced apoptotic effects in cisplatin-resistant HeLa cells were mediated via cell cycle arrest, ROS production and targeting of mTOR/PI3K/AKT signalling pathway. Thus, tormentic acid may be considered as a lead molecule in cancer therapeutics.