Vitex Rotundifolia fractions induced apoptosis in human breast cancer T-47d cell line via activation of extrinsic and intrinsic pathway

Abstract

Objective: Breast cancer is the most frequently diagnosed cancer worldwide. The main objective of the present study was to evaluate the cytotoxic effects and mechanism of cell death induced by the extract and fractions of Vitex rotundifolia (leaves) in breast cancer cell line, T-47D. Methods: The cytotoxicity activity was measured using MTS assay. The mode of cell death was analysed by early (phosphatidylserine externalization) and late apoptosis (DNA fragmentation). The caspases 8, 9, 3/7 and apoptotic proteins bax, bcl-2 study were done by western blot and ELISA method. Results: The methanol extract was found to inhibit 50% growth of T-47D cells at the concentration of 79.43μg/ml respectively after 72hr. From seven fractions, fraction F1, F2 and F3 produced cytotoxicity effects in T-47D cell line with IC50 (72hr) < 30μg/ml. The results obtained by Annexin V/PI apoptosis detection assay and TUNEL assay suggest that active fractions of Vitex rotundifolia induced early and late apoptosis (DNA fragmentation) in T-47D cell line. Moreover, western blot analysis and Caspase GloTM luminescent assay demonstrated that fractions F2 and F3 triggered apoptotic cell death via activation of caspases -8, -9 and -3/7 and up-regulation of Bax and down-regulation of Bcl-2 protein. Furthermore, chemical profiling confirms the presence of potential metabolites (vitexicarpin) in fractions of Vitex rotundifolia. Conclusion: Thus, the present study suggests the remarkable potential of active metabolites in fractions of Vitex rotundifolia as future cancer therapeutic agent for the treatment of breast cancer.