Triptolide Induces Glioma Cell Autophagy and Apoptosis via Upregulating the ROS/JNK and Downregulating the Akt/mTOR Signaling Pathways.
ABSTRACT: Apoptosis and autophagy are the two prominent forms of developmental cell death, and researches have shown that crosstalk exists between these two processes. A prior study demonstrated that triptolide inhibited the proliferation of malignant glioma cells. However, whether apoptosis and autophagy participate in the inhibitory effect of triptolide in glioma cells has not been clarified. In the present study, we demonstrated that triptolide potently inhibited the growth of glioma cells by inducing cell cycle arrest at the G2/M phase. Additionally, the treatment with triptolide induced apoptosis and autophagy in various glioma cell lines. Triptolide-induced autophagy may have tumor-supporting effects. Autophagy and apoptosis could cross-inhibit each other in glioma cells treated with triptolide. Moreover, we found that triptolide induced ROS production and JNK activation and inhibited the activity of Akt and mTOR. Finally, we demonstrated that triptolide suppressed tumor growth in an orthotopic xenograft glioma model. Collectively, these data indicated that triptolide induced G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK and blocking the Akt/mTOR signaling pathways in glioma cells. Triptolide may be a potential anti-tumor drug targeting gliomas.
Project description:<h4>Background</h4>Celastrol, a triterpene compound derived from the traditional Chinese medicine Tripterygium wilfordii, has been reported to possess potential antitumor activity towards various malignancies. However, the effect of celastrol on glioma cells and the underlying molecular mechanisms remain elusive.<h4>Methods</h4>Glioma cells, including the U251, U87-MG and C6 cell lines and an animal model were used. The effects of celastrol on cells were evaluated by flow cytometry, confocal microscopy, reactive oxygen species production assay and immunoblotting after treatment of celastrol. Fisher's exact test, a one-way ANOVA and the Mann-Whitney U-test were used to compare differences between groups. All data were analyzed using SPSS version 21.0 software.<h4>Results</h4>Here, we found that exposure to celastrol induced G2/M phase arrest and apoptosis. Celastrol increased the formation of autophagosomes, accumulation of LC3B and the expression of p62 protein. Celastrol-treated glioma cells exhibited decreased cell viability after the use of autophagy inhibitors. Additionally, autophagy and apoptosis caused by celastrol in glioma cells inhibited each other. Furthermore, celastrol induced JNK activation and ROS production and inhibited the activities of Akt and mTOR kinases. JNK and ROS inhibitors significantly attenuated celastrol-trigged apoptosis and autophagy, while Akt and mTOR inhibitors had opposite effects.<h4>Conclusions</h4>In conclusion, our study revealed that celastrol caused G2/M phase arrest and trigged apoptosis and autophagy by activating ROS/JNK signaling and blocking the Akt/mTOR signaling pathway.
Project description:Human bladder cancer is a common genitourinary malignant cancer worldwide. However, new therapeutic strategies are required to overcome its stagnated survival rate. Triterpene glycoside Actein (ACT), extracted from the herb black cohosh, suppresses the growth of human breast cancer cells. Our study attempted to explore the role of ACT in human bladder cancer cell growth and to reveal the underlying molecular mechanisms. We found that ACT significantly impeded the bladder cancer cell proliferation via induction of G2/M cycle arrest. Additionally, ACT administration triggered autophagy and apoptosis in bladder cancer cells, proved by the autophagosome formation, LC3B-II accumulation, improved cleavage of Caspases/poly (ADP-ribose) polymerase (PARP). Furthermore, reduction of reactive oxygen species (ROS) and p-c-Jun N-terminal kinase (JNK) could markedly reverse ACT-induced autophagy and apoptosis. In contrast, AKT and mammalian target of rapamycin (mTOR) were greatly de-phosphorylated by ACT, while suppressing AKT and mTOR activity could enhance the effects of ACT on apoptosis and autophagy induction. In vivo, ACT reduced the tumor growth with little toxicity. Taken together, our findings indicated that ACT suppressed cell proliferation, induced autophagy and apoptosis through promoting ROS/JNK activation, and blunting AKT pathway in human bladder cancer, which indicated that ACT might be an effective candidate against human bladder cancer in future.
Project description: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.
Project description:Physagulide P (PP), a new natural compound, was isolated from Physalis angulate L. in our laboratory. In this study, we demonstrated that PP potently suppressed cell proliferation by inducing G2/M phase arrest in MDA-MB-231 and MDA-MB-468 cells. Moreover, PP provoked apoptosis by decreasing the mitochondrial membrane potential and elevating the Bax/Bcl-2 protein expression ratio. The caspase inhibitor Z-VAD-FMK partly restore cell viability, suggesting that apoptosis plays as an important role in the anti-proliferative effect of PP. PP-treated cells also underwent autophagy, as evidenced by the formation of autophagosomes and the accumulation of LC3BII. Furthermore, the knockdown of LC3B reduced PP-induced cytotoxicity, indicating that autophagy played an anticancer effect. PP also induced the generation of reactive oxygen species (ROS) and resulted in c-Jun N-terminal kinases (JNK) activation. Accordingly, JNK siRNA significantly attenuated PP-triggered apoptosis and autophagy, and ROS scavengers almost completely reverse this apoptosis and autophagy. The ROS scavenger also blocked PP-induced G2/M phase arrest and the phosphorylation of JNK. Our results revealed that PP induced G2/M phase arrest, apoptosis and autophagy via the ROS/JNK signaling pathway in MDA-MB-231 and MDA-MB-468 cells. Therefore, PP is a promising candidate for the development of antitumor drugs for the treatment of triple-negative breast cancer.
Project description:Purpose:Arctigenin (ARG) is a natural lignan compound extracted from Arctium lappa and has displayed anticancer function and therapeutic effect in a variety of cancers. Arctigenin is mainly from Arctium lappa extract. It has been shown to induce autophagy in various cancers. However, as for whether arctigenin induces autophagy in gliomas or not, the specific mechanism is still worth exploring. Methods:Using CCK8, the monoclonal experiment was made to detect the proliferation ability. The scratch experiment and the transwell experiment were applied to the migration and invasion ability. PI/RNase and FITC-conjugated anti-annexin V were used to detect the cell cycle and apoptosis. Western blotting was used to determine the specified protein level, and constructed LC3B-GFP plasmid was used for analysis of autophagy. Results:Our research showed that ARG inhibited the growth and proliferation and invasion and migration of glioma cells in a dose-dependent manner (U87MG and T98G) and arrested the cell cycle and induced apoptosis. Interestingly, ARG induced autophagy in a dose-dependent manner. We applied Western blotting to measure the increase in the key autophagy protein LC3B, as well as some other autophagy-related proteins (increase in Beclin-1 and decrease in P62). In order to further explore the mechanism that ARG passed initiating autophagy to inhibit cell growth, we further found by Western blotting that AKT and mTOR phosphorylation proteins (P-AKT, P-mTOR) were reduced after ARG treatment, and we used AKT agonists to rescue, and the phosphorylated proteins of AKT and mTOR increased, and we found that the autophagy-related proteins were also reversed. And interestingly, the protein of apoptosis was also reversed along with autophagy. Conclusions:We thought ARG inhibited the proliferation of glioma cells by inducing autophagy and apoptosis through the AKT/mTOR pathway.
Project description:Triptolide, an active compound extracted from the Chinese herb thunder god vine (Tripterygium wilfordii Hook F.), has potent anti-tumor activity. Recently, triptolide was found to induce autophagy in cancer cells. However, the effects of triptolide on autophagy in human prostate cancer (PCa) cells have not yet been clearly elucidated. In this study, we demonstrated that triptolide induces autophagy in three PCa cell lines, PC-3, LNCaP and C4-2. Furthermore, we found that triptolide mediates intracellular accumulation of free calcium by stimulating the endoplasmic reticulum (ER) stress response. This activates the CaMKK?-AMPK signaling pathway, which in turn inhibits mTOR and activates both ULK1 and Beclin 1, finally resulting in autophagy. Moreover, we found that treatment with autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhances triptolide-induced PCa cell death and growth inhibition. Using a PC-3-xenografted mouse model, we showed that blocking autophagy with CQ significantly promoted triptolide-induced tumor growth inhibition in vivo. Overall, our results show that triptolide induces protective autophagy through the CaMKK?-AMPK pathway in PCa cells, implying that a combination of triptolide with autophagy inhibitors may potentially be an effective therapeutic strategy for PCa.
Project description:Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5'-triphosphate-competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.
Project description:Oncolytic adenoviruses, such as Delta-24-RGD (?24RGD), are replication-competent viruses that are genetically engineered to induce selective cancer cell lysis. In cancer cells, ?24RGD induces massive autophagy, which is required for efficient cell lysis and adenoviral spread. Understanding the cellular mechanisms underlying the regulation of autophagy in cells treated with oncolytic adenoviruses may provide new avenues to improve the therapeutic effect. In this work, we showed that cancer cells infected with ?24RGDundergo autophagy despite the concurrent activation of the AKT/mTOR pathway. Moreover, adenovirus replication induced sustained activation of JNK proteins in vitro. ERK1/2 phosphorylation remained unchanged during adenoviral infection, suggesting specificity of JNK activation. Using genetic ablation and pharmacological inactivation of JNK, we unequivocally demonstrated that cells infected with ?24RGD required JNK activation. Thus, genetic co-ablation of JNK1 and JNK2 genes or inhibition of JNK kinase function rendered ?24RGD-treated cells resistant to autophagy. Accordingly, JNK activation induced phosphorylation of Bcl-2 and prevented the formation of Bcl-2/Beclin 1 autophagy suppressor complexes. Using an orthotopic model of human glioma xenograft, we showed that treatment with ?24RGD induced phosphorylation and nuclear translocation of JNK, as well as phosphorylation of Bcl-2. Collectively, our data identified JNK proteins as an essential mechanistic link between ?24RGD infection and autophagy in cancer cells. Activation of JNK without inactivation of the AKT/mTOR pathway constitutes a distinct molecular signature of autophagy regulation that differentiates ?24RGD adenovirus from the mechanism used by other oncolytic viruses to induce autophagy and provides a new rationale for the combination of oncolytic viruses and chemotherapy.
Project description:BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive of the primary brain tumors, with a grim prognosis despite intensive treatment. In the past decades, progress in research has not significantly increased overall survival rate. METHODS: The in vitro antineoplastic effect and mechanism of action of Casiopeina III-ia (Cas III-ia), a copper compound, on rat malignant glioma C6 cells was investigated. RESULTS: Cas III-ia significantly inhibited cell proliferation, inducing autophagy and apoptosis, which correlated with the formation of autophagic vacuoles, overexpression of LC3, Beclin 1, Atg 7, Bax and Bid proteins. A decrease was detected in the mitochondrial membrane potential and in the activity of caspase 3 and 8, together with the generation of intracellular reactive oxygen species (ROS) and increased activity of c-jun NH(2)-terminal kinase (JNK). The presence of 3-methyladenine (as selective autophagy inhibitor) increased the antineoplastic effect of Cas III-ia, while Z-VAD-FMK only showed partial protection from the antineoplastic effect induced by Cas III-ia, and ROS antioxidants (N-acetylcysteine) decreased apoptosis, autophagy and JNK activity. Moreover, the JNK -specific inhibitor SP600125 prevented Cas III-ia-induced cell death. CONCLUSIONS: Our data suggest that Cas III-ia induces cell death by autophagy and apoptosis, in part due to the activation of ROS -dependent JNK signaling. These findings support further studies of Cas III-ia as candidate for treatment of human malignant glioma.
Project description:Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.