Celastrol induces apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells: an in vitro and in vivo study

Osteosarcoma is the most common primary malignant tumor of bone, the long-term survival of which has stagnated in the past several decades. Celastrol, a triterpene from traditional Chinese medicine, has been proved to possess potent anti-tumor effect on various cancers. However, the effect of celastrol on human osteosarcoma and the underlying mechanisms remains to be elucidated. 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. Furthermore, inhibition of apoptosis enhanced autophagy while suppression of autophagy diminished apoptosis. Celastrol also induced JNK activation and ROS generation. The JNK inhibitor significantly attenuated celastrol-triggered apoptosis and autophagy while ROS scavenger could completely reverse them. The ROS scavenger also prevented G2/M phase arrest and phosphorylation of JNK. Importantly, we found that celastrol had the similar effects on primary osteosarcoma cells. Finally, in vivo, celastrol suppressed tumor growth in the mouse xenograft model. 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.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.¹ The 5-year survival of patients with localized osteosarcoma has improved to 60% due to the multi-agent, dose-intensive chemotherapy in conjunction with gradually improved surgical techniques, but has remained largely unchanged during the last three decades.² At the same time, the high-dose use of chemotherapeutic drugs is limited due to their systemic toxicity. 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.^{3, 4, 5} Recently, celastrol has attracted great attention for its potent anticancer effects and its diverse molecular targets involved in tumorigenesis have been reported.^{6, 7, 8, 9, 10, 11, 12} Although these molecular targets have positive correlations with inhibition of tumors, it is not clear which, if any, is the direct target or the principal mediator. Meanwhile, whether celastrol suppresses the growth of human osteosarcoma has never been investigated before.Cell cycle deregulation is a hallmark of tumor cells and defective checkpoint function results in genetic modifications that contribute to tumorigenesis.¹³ The G2/M checkpoint is one of the conspicuous targets for anticancer drugs. The cyclin B1/ Cdc2 complex, which plays a key role in promoting the G2/M phase transition, is regulated by a range of proteins, including Cdc2, Cdc25C, Chk1/2 and p21.^{14, 15, 16}Cell death could be classified into apoptosis, autophagy, necrosis, cornification and tentative definitions of atypical cell death modalities.¹⁷ Apoptosis, as the type-I programmed cell death (PCD), plays a key role in chemotherapies against a variety of cancers.¹⁸ Autophagy is regarded as type-II PCD and the caspase-independent cell death pathway.¹⁹ In some cellular settings, autophagy serves as a cell survival pathway, suppressing apoptosis, and, in others, it can lead to cell death itself, either in collaboration with apoptosis or as a back-up mechanism when the former is defective.²⁰ Whether celastrol can induce apoptosis or autophagy, what roles do they have and the interplay between each other in celastrol-induced cell death of osteosarcoma remain to be determined.Reactive oxygen species (ROS), active forms of oxygen, generate as by-products from cellular metabolism.²¹ A moderate increase in ROS can promote cell proliferation and differentiation, whereas excessive amounts of ROS can interfere with cellular signaling pathways by causing oxidative damage to lipids, proteins and DNA.^{22, 23, 24} Interestingly, accumulating evidence suggests that cancer cells are under increased oxidative stress, and therefore more vulnerable to damage by further ROS insults induced by exogenous agents.²⁵ Furthermore, ROS could affect various signaling pathways such as MAPK signal transduction cascades.^{26, 27} As a stress-activated protein kinase (SAPK) of the MAPK family, JNK plays a pivotal role in many cellular events, including apoptosis and autophagy.^{28, 29} Accordingly, targeted inhibition of related signaling pathways, particularly the ROS/JNK signaling, may be effective in the treatment of human cancers.In the present study, we elucidated the inhibitory effect of celastrol on osteosarcoma cell lines and primary cells in vitro and in vivo. We further explored the molecular mechanisms, that is, induction of G2/M phase arrest, apoptosis and autophagy mediated by the ROS/JNK signaling pathway.