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

ABSTRACT: 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.

SUBMITTER: Li HY

PROVIDER: S-EPMC4669742 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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

Li H-Y HY Zhang J J Sun L-L LL Li B-H BH Gao H-L HL Xie T T Zhang N N Ye Z-M ZM

Cell death & disease 20150122

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 r ...[more]

PMID: 25611379

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Project description:Aims: Peiminine has been reported to have various pharmacological properties, including anticancer activity. In this study, we investigated the effect of this alkaloid on osteosarcoma and explored the underlying mechanisms. Methods: To evaluate the antiosteosarcoma effects of peiminine in vitro, cell viability was assessed by CCK-8 and live/dead assays; the effects of the drug on apoptosis and the cell cycle were examined by flow cytometry; the effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively, while its effects on autophagy were observed by transmission electron microscopy and an LC3 fluorescent puncta formation assay. The role of autophagy in the peiminine-mediated effects in osteosarcoma cells was evaluated by CCK-8 assay and western blotting after the application of the autophagy inhibitor chloroquine. The effect of peiminine on reactive oxygen species (ROS) production was analyzed using fluorescence confocal microscopy and spectrophotometry. Additionally, peiminine-treated osteosarcoma cells were exposed to SP600125, a JNK inhibitor, and N-acetylcysteine, a ROS scavenger, after which the contribution of the ROS/JNK signaling pathway to osteosarcoma was assessed using cell viability and LC3 fluorescent puncta formation assays, flow cytometry, and western blotting. A xenograft mouse model of osteosarcoma was generated to determine the antitumor effects of peiminine in vivo. Results: Peiminine suppressed proliferation and metastasis and induced cell cycle arrest, apoptosis, and autophagy in osteosarcoma cells. These anticancer effects of peiminine were found to be dependent on intracellular ROS generation and activation of the JNK pathway. In line with these results, peiminine significantly inhibited xenograft tumor growth in vivo. Conclusions: Peiminine induced G0/G1-phase arrest, apoptosis, and autophagy in human osteosarcoma cells via the ROS/JNK signaling pathway both in vitro and in vivo. Our study may provide an experimental basis for the evaluation of peiminine as an alternative drug for the treatment of osteosarcoma.

Project description:Celastrol, a plant-derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species (ROS). Recently, we have reported that cadmium (Cd) activates c-Jun N-terminal kinase (JNK) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 (PP5), and celastrol prevents Cd-activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS-dependent PP5-JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd-induced expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22phox , p40phox , p47phox , p67phox , and Rac1), as well as the generation of ROS in PC12 cells and primary neurons. Also, N-acetyl-l-cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd-evoked NOX2-derived ROS. Further research revealed that celastrol was involved in the regulation of PP5 inactivation and JNK/c-Jun activation induced by Cd, as celastrol prevented Cd from reducing PP5 expression, and over-expression of wild-type PP5 or dominant negative c-Jun strengthened celastrol's inhibition of Cd-induced phosphorylation of JNK and/or c-Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX2 with apocynin or silencing NOX2 by RNA interference enhanced the inhibitory effects of celastrol on Cd-induced inactivation of PP5, activation of JNK/c-Jun, ROS, and apoptosis in the cells. Furthermore, we noticed that expression of wild-type PP5 or dominant negative c-Jun, or pretreatment with JNK inhibitor SP600125 reinforced celastrol's suppression of Cd-induced NOX2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd-induced neuronal apoptosis via targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Dataset Information

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

Publications

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

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