Withaferin-A induces apoptosis in osteosarcoma U2OS cell line via generation of ROS and disruption of mitochondrial membrane potential

Background: Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania sominifera. It has tremendous pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis inducing drug-like molecules. Osteosarcoma is a rare type of osteocancer, affecting human. The present study therefore focused on the evaluation of antitumor potential of WF-A against several osteosarcoma cell lines. Materials and Methods: MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC₅₀. DAPI staining was used to confirm the apoptosis inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. Results: The results revealed that that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC₅₀ ranging from 0.32 to 7.6 μM. The lowest IC₅₀ (0.32 μM) was observed against U2OS cell line and therefore it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced ROS-mediated reduction in mitochondrial membrane potential ΔΨm) in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. Conclusion: We propose that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential.