Celastrol induces cycle arrest, apoptosis and autophagy via ROS/JNK pathway in osteosarcoma cells

Abstract

Objective To explore the effect of celastrol on human osteosarcoma and the underlying mechanisms. Methods Osteosarcoma cell lines(HOS, MG-63, U-2OS and Saos-2)and primary cells were used for test. The anti-proliferative effect of celastrol on osteosarcoma cells was determined by the MTS kit and clone formation assay. Cell cycle was monitored by flow cytometry and Western Blot for related cell cycle proteins. Hoechst staining, transmission electron microscopy, flow cytometry and Western Blot were used for detection of apoptosis and autophagy induced by celastrol. The level of ROS and JNK were investigated by fluorescent probe and Western Blot assay. Meanwhile, corresponding inhibitors of apoptosis(z-VAD), autophagy(3-MA), ROS(NAC)and JNK(SP600125)were added for confirming the phenomenon above. Finally, osteosarcoma xenograft experiment was performed, the result of which was determined by Western Blot assay. Results Celastrol could potently inhibit cell proliferation of osteosarcoma by causing G2/M phase arrest. Exposure to celastrol resulted in the activation of caspase-3,-8, and-9, indicating that celastrol induced apoptosis through both extrinsic and intrinsic pathways. Autophagy also occurred in celastrol-treated cells as evidenced by formation of autophagosome and accumulation of LC3B-II. There exist complex interplays between apoptosis and autophagy. Inhibition of apoptosis enhanced autophagy while suppression of autophagy diminished apoptosis. The celastrol-induced cell death was remarkably restored by the combination of autophagy and apoptosis inhibitors. Celastrol also induced JNK activation and ROS generation. JNK inhibitor significantly attenuated celastrol-triggered apoptosis and autophagy while ROS scavenger could completely reverse them. ROS scavenger also prevented G2/M phase arrest and phosphorylation of JNK. Importantly, celastrol had the similar effects on primary osteosarcoma cells and suppressed tumor growth in the mouse xenograft model with sound safety. Conclusion Celastrol caused G2/M phase arrest, induced apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells. Key words: Tripterygium wilfordii; Osteosarcoma; Apoptosis; Autophagy; Reactive oxygen species