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Essential role of mitochondrial Stat3 in p38MAPK mediated apoptosis under oxidative stress.

ABSTRACT: Stat3 is an oncogene, frequently associated with malignant transformation. A body of evidence implicates that phospho-Stat3Y705 contributes to its nucleic translocation, while phospho-Stat3S727 leads to the accumulation in mitochondria. Both are of importance for tumor cell proliferation. In comparison to well-characterized signaling pathways interplaying with Stat3Y705, little is known about Stat3S727. In this work, we studied the influence of Stat3 deficiency on the viability of cells exposed to H2O2 or hypoxia using siRNA and CRISPR/Cas9 genome-editing. We found dysregulation of mitochondrial activity, which was associated with excessive ROS formation and reduced mitochondrial membrane potential, and observed a synergistic effect for oxidative stress-mediated apoptosis in Stat3-KD cells or cells carrying Stat3Y705F, but not Stat3S727D, suggesting the importance of functional mitochondrial Stat3 in this context. We also found that ROS-mediated activation of ASK1/p38MAPK was involved and adding antioxidants, p38MAPK inhibitor, or genetic repression of ASK1 could easily rescue the cellular damage. Our finding reveals a new role of mitochondrial Stat3 in preventing ASK1/p38MAPK-mediated apoptosis, wich further support the notion that selective inhibition mitochondrial Stat3 could provide a primsing target for chemotherapy.

SUBMITTER: Cheng X

PROVIDER: S-EPMC5684365 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Essential role of mitochondrial Stat3 in p38MAPK mediated apoptosis under oxidative stress.

Cheng Xinlai X Peuckert Christiane C Wölfl Stefan S

Scientific reports 20171113 1

Stat3 is an oncogene, frequently associated with malignant transformation. A body of evidence implicates that phospho-Stat3Y705 contributes to its nucleic translocation, while phospho-Stat3S727 leads to the accumulation in mitochondria. Both are of importance for tumor cell proliferation. In comparison to well-characterized signaling pathways interplaying with Stat3Y705, little is known about Stat3S727. In this work, we studied the influence of Stat3 d ...[more]

PMID: 29133922

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Project description:In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome-lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome-lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome-lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)-GFP-LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER-Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome-lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome-lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy.

Project description:We evaluated the mechanism of capsaicin-mediated ROS generation in pancreatic cancer cells. The generation of ROS was about 4-6 fold more as compared to control and as early as 1 h after capsaicin treatment in BxPC-3 and AsPC-1 cells but not in normal HPDE-6 cells. The generation of ROS was inhibited by catalase and EUK-134. To delineate the mechanism of ROS generation, enzymatic activities of mitochondrial complex-I and complex-III were determined in the pure mitochondria. Our results shows that capsaicin inhibits about 2.5-9% and 5-20% of complex-I activity and 8-75% of complex-III activity in BxPC-3 and AsPC-1 cells respectively, which was attenuable by SOD, catalase and EUK-134. On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells. The ATP levels were drastically suppressed by capsaicin treatment in both BxPC-3 and AsPC-1 cells and attenuated by catalase or EUK-134. Oxidation of mitochondria-specific cardiolipin was substantially higher in capsaicin treated cells. BxPC-3 derived ?(0) cells, which lack mitochondrial DNA, were completely resistant to capsaicin mediated ROS generation and apoptosis. Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells. Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level. Over-expression of catalase by transient transfection protected the cells from capsaicin-mediated ROS generation and apoptosis. Furthermore, tumors from mice orally fed with 2.5 mg/kg capsaicin show decreased SOD activity and an increase in GSSG/GSH levels as compared to controls. Taken together, our results suggest the involvement of mitochondrial complex-I and III in capsaicin-mediated ROS generation and decrease in antioxidant levels resulting in severe mitochondrial damage leading to apoptosis in pancreatic cancer cells.

Dataset Information

Essential role of mitochondrial Stat3 in p38MAPK mediated apoptosis under oxidative stress.

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Essential role of mitochondrial Stat3 in p38<sup>MAPK</sup> mediated apoptosis under oxidative stress.

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