Celastrol Induces Apoptosis And Autophagy Via ROS/JNK Pathway In Osteosarcoma Cells

Osteosarcoma is the most common primary malignant tumor of bone, occurring predominantly in children and adolescents with a very high propensity for local invasion and early systemic metastases. The5-year survival of patients with localized osteosarcoma has improved to60-80%due to the multi-agent, dose-intensive chemotherapy in conjunction with gradually-improved surgical techniques in the early1980s, but has remained largely unchanged during the last three decades. By contrast, in the metastatic and recurrent conditions, the long-term survival of osteosarcoma patients still remains at10-30%. At the same time, the high-dose use of chemotherapeutic drugs (methotrexate, doxorubicin, cisplatin and ifosfamide) is limited due to their systemic toxicity, such as nephrotoxicity and neurotoxicity. Therefore, the development of novel therapies for the management of osteosarcoma is especially urgent.Celastrol, a triterpenoid isolated from the traditional Chinese medicine "Thunder of God Vine", has been used in the treatment of autoimmune and neurodegenerative diseases. In recent years, celastrol has attracted great attention for its potent anticancer effects in vitro and in vivo. Diverse molecular targets involved in tumorigenesis modulated by celastrol have been reported, including NF-κB, heat shock protein90(HSP90), proteasome, MAPK, STAT3. Although these molecular targets have positive correlations with celastrol-induced inhibition of tumors, it is not clear which, if any, is the direct target or the principal mediator. Meanwhile, whether celastrol exerts its anticancer effect on human osteosarcoma has never been investigated before. We reported here that celastrol could inhibit cell proliferation 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 occurred in celastrol-treated cells as evidenced by formation of autophagosome and accumulation of LC3B-Ⅱ. 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, indicating that ROS most likely acts as an initiator in celastrol-induced effects.We obtained different types of primary cells from osteosarcoma patients, including cases of metastasis, recurrence, insensitive or multidrug resistant to chemotherapy. We found that celastrol had the similar effects on primary osteosarcoma cells.Finally, in vivo, celastrol at doses of1and2mg/kg significantly inhibited tumor growth in the mouse xenograft model during the7-day treatment period. Western blot and immunohistochemical analysis of tumor tissues confirmed the similar effects in intro.Taken together, our results revealed that celastrol caused G2/M phase arrest, induced apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells. Celastrol is therefore a promising candidate for development of antitumor drugs targeting osteosarcoma.