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Blocking autophagy enhances the apoptotic effect of 18β-glycyrrhetinic acid on human sarcoma cells via endoplasmic reticulum stress and JNK activation.

ABSTRACT: Sarcoma, a rare form of cancer, is unlike the much more common carcinomas as it occurs in a distinct type of tissue. The potent antitumor effects of 18β-glycyrrhetinic acid (GA), a novel naturally derived agent, have been demonstrated in various cancers. However, the effect of GA on human sarcoma, and the underlying mechanisms, remain to be elucidated. In the current study, we show that GA inhibits sarcoma cell proliferation by inducing G0/G1-phase arrest. Exposure to GA resulted in the activation of caspase-3, -8, and -9, indicating that GA induced apoptosis through both extrinsic and intrinsic pathways. In addition, the autophagy pathway, characterized by the conversion of LC3-I to LC3- II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression, and stimulation of autophagic flux. The present findings showed that GA stimulated autophagy by inducing endoplasmic reticulum (ER) stress via the IRE1-JNK pathway. Our data supported the prosurvival role of GA-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors. Finally, GA markedly reduced sarcoma growth, with little organ-related toxicity, in vivo. The present results suggest that the combination of GA with a specific autophagy inhibitor represents a promising therapeutic approach for the treatment of sarcoma.

SUBMITTER: Shen S

PROVIDER: S-EPMC5636985 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundXanthoangelol (XAG) was reported to exhibit antitumor properties in several cancer. However, the specific anti-tumor activity of XAG in human hepatocellular carcinoma (HCC) and the relevant mechanisms are not known.MethodsThe effects of XAG on HCC cell proliferation and apoptosis were respectively examined by CCK-8 assay and Annexin V-FITC/PI apoptosis kit. Western blotting was conducted to detect the expression of proteins. The effect of XAG on the development of acidic vesicle organelles was assessed using acridine orange staining. mRFP-GFP-LC3 adenovirus was used to transfect HCC cells and the formation of autolysosome was detected using a confocal microscope.ResultsMechanistically, XAG promotes HCC cell death through triggering intrinsic apoptosis pathway, not extrinsic apoptotic pathway. Furthermore, XAG treatment induced autophagy in Bel 7402 and SMMC 7721 cells, as evidenced by an increase in autophagy-associated proteins, including LC3B-II, Beclin-1, and Atg5. Interestingly, inhibition of autophagy with 3-MA, Bafilomycin A1 (Baf A1), or siRNA targeting Atg5 effectively enhanced the apoptotic cell ratio in XAG-treated cells, indicating that protective effect of autophagy induced by XAG in HCC. Moreover, autophagy induced by XAG was mediated by activating endoplasmic reticulum stress (ERS), along with administration of XAG, the expression levels of ERS-associated proteins, including CHOP, GRP78, ATF6, p-eIF2α, IRE1α, and cleaved caspase-12 were significantly increased in HCC cells. Meanwhile, suppressing ERS with chemical chaperones (TUDCA) or CHOP shRNA could effectively abrogate the autophagy-inducing effect of XAG, and increase the apoptotic cell death. Further mechanistic studies showed that ERS-induced autophagy in XAG-treated cells was mediated by activation of JNK/c-jun pathway. XAG treatment resulted in the increase of p-JNK and p-c-jun, while suppressing ERS with TUDCA or CHOP shRNA could effectively reverse it. Meanwhile, SP600125, a JNK inhibitor, effectively reversed XAG-induced protective autophagy and enhanced cell apoptosis in XAG-treated HCC cells. In vivo results demonstrated that XAG exerts potent antitumor properties with low toxicity.ConclusionsCollectively, these results suggested that XAG could be served as a promising candidate for the treatment and prevention of HCC.

Project description:BackgroundEndoplasmic reticulum (ER) stress and its consequent unfolded protein response (UPR) are believed to be associated with progression, survival and chemoresistance of a variety of tumor cells through multiple cellular processes, including autophagy. Therefore, the ER stress-autophagy pathway presents a potential molecular target for therapeutic intervention. The objective of this study was to evaluate the therapeutic efficacy of ER stress and autophagy modulators in the context of pancreatic ductal adenocarcinoma (PDAC).MethodsWe first targeted IRE1?, an important regulator of the UPR, through STF-083010 treatment in PDAC cell lines in vitro. Chloroquine was then used to target autophagy and an optimal combination treatment was developed using chloroquine, sunitinib and gemcitabine. Apoptosis was analyzed using TUNEL assay, autophagy was estimated using lysotracker staining and electron microscopy, and UPR was analyzed using anti-GRP78 immunostaining and XBP1 splicing. Transplantation of PDAC derived KPCP1 and Panc02 cells in mouse pancreas were performed to study treatment efficacy in vivo.ResultsSuppression of the IRE1? by STF-083010 alone resulted in increased lysosomes and reduced viability of PDAC cells. Chloroquine treatment alone inhibited downstream autophagy but was insufficient in reducing PDAC cell growth. However, combining STF-083010 and chloroquine had additive anti-tumor efficacy when used with gemcitabine. Sunitinib alone caused abnormal maturation of the autolysosomes with increased intracellular multivesicular bodies and increased apoptosis evident in PDAC cells. Sunitinib showed a synergistic effect with chloroquine in reducing in vitro PDAC cell viability and significantly increased the efficacy of gemcitabine in human and murine PDAC cell lines. The anti-proliferative effect of gemcitabine was significantly increased when used in combination with sunitinib and/or chloroquine in both in vitro and in vivo PDAC models. The addition of sunitinib and/or chloroquine to gemcitabine, resulted in a significantly increased survival of the animals without noticeably increased toxicity. Sunitinib, gemcitabine and chloroquine treated mice showed a significant reduction of GRP78 expression, reduced cell proliferation and increased apoptosis in pancreas, compatible with a tumor response.ConclusionsSunitinib combined with chloroquine reduces tumor growth through suppression of autophagy and increased apoptosis. Co-administration of modulators of ER stress-mediated autophagy with chemotherapy presents a novel therapeutic approach in PDAC.

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Blocking autophagy enhances the apoptotic effect of 18β-glycyrrhetinic acid on human sarcoma cells via endoplasmic reticulum stress and JNK activation.

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