OBJECTIVE Glioblastomas(GBM) are the most malignant brain tumors in humans and have a very poor prognosis.New therapeutics are urgently needed.Here,we reported 2-methoxy-6-acetyl-7-methyljuglone(MAM)-induced cell deat...OBJECTIVE Glioblastomas(GBM) are the most malignant brain tumors in humans and have a very poor prognosis.New therapeutics are urgently needed.Here,we reported 2-methoxy-6-acetyl-7-methyljuglone(MAM)-induced cell death in U87 and U251 glioma cancer cells.METHODS Cells were cultured and treated with MAM,the cell viability was determined by MTT assay and LDH assay.Intracellular reactive oxygen species(ROS) generation was observed by DCF fluorescence.The protein expression was determined by Western blotting.RESULTS MAM induced glioma cancer cell death without caspase activation.The cell death induced by MAM was attenuated by the pharmacological or genetic blockage of necroptosis signaling,including RIP1 inhibitor necrostatin-1 s(Nec-1 s) and siRNA-mediated gene silencing of RIP1 and RIP3,but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1 H-Indol-3-yl)-3-pentylamino-maleimide(IM54).MAM treated U87 and U251 glioma cancer cells induced RIP1/RIP3 complex formation,ROS level increased,ATP concentration decreased and loss of plasma membrane integrity,further confirmed this process was necroptosis.The essential role of ROS was confirmed by the protective effect of ROS scavenger NAC.Interestingly,MAM induced necroptosis both triggered by RIP1/RIP3 complex and ROS generation.Moreover,MAM induced necroptosis through cytosolic calcium(Ca2 +) accumulation and sustained c-Jun N-terminal kinase(JNK) activation.Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate cell death.Further,we found there exists a feedback loop between RIP1 and JNK activation.Finally,MAM induced necroptosis was inhibited by dicoumarol(a NQO1 inhibitor).Dicoumarol exposed glioma cancer cells were resistant to RIP1/RIP3 complex formation and ROS generation.MAM induced necroptosis was independent of MLKL.CONCLUSION MAM induced non-canonical necroptosis through the NQO1-dependent ROS and RIP1/RIP3 pathway.This study also provided new insights into the molecular regulation of necroptosis in human glioma cancer cells and a promising approach for GBM treatment.展开更多
OBJECTIVE To investigate the mechanism of anticancer effect of 2-methoxystypandrone(2-MS),a natural naphthoquinone isolated from Polygonum cuspidatum Sieb.et Zucc.METHODSThree types of cancer cells were investigated i...OBJECTIVE To investigate the mechanism of anticancer effect of 2-methoxystypandrone(2-MS),a natural naphthoquinone isolated from Polygonum cuspidatum Sieb.et Zucc.METHODSThree types of cancer cells were investigated in the research(A549,MCF7,B16-F10).Flow cytometer was used to determine ROS/RNS generation.Western blotting was used to detect related protein expression.Apoptosis assay,GSH/GSSG(reduced glutathione/oxidized glutathione)assay were performed using commercial kit.SiRNA knockdown was used to silence cj-un N-terminal kinase(JNK)and iNOS.High-performance liquid chromatography(HPLC)was used to detect the direct reaction of 2-MS with GSH.RESULTS 2-MS induced cytotoxity towards a panel of cancer cells,with less effect on normal cells.2-MS induced necroptosis in A549 cell and apoptosis in B16-F10 and MCF7cells.2-MS increased phosphorylation of JNK in three types of cancer cells.Inhibition of JNK with SP600125 or silencing JNK attenuated 2-MS-induced cell death.JNK also activated iNOS expression and led to nitric oxide(NO)generation in three cancer cells.NO-induced nitrative stress was responsible for DNA damage and necroptosis in A549 cells.NO also inhibited NF-κB expression and induced intrinsic apoptosis in B16-F10 and MCF7cells.Both NO scavenger hemoglobin and silencing iNOS can partially reverse 2-MS-induced cell death.Furthermore,we found that all of these were attributed to induction of hydrogen peroxide(H2O2),which was caused by glutathione(GSH)depletion through interaction of 2-MS with GSH.The interaction was validated through cell-free HPLC analysis.Both the H2O2 scavenger catalase and exogenous GSH can significantly reverse the 2-MS-induced cell death.But catalase did not protect against the decrease in GSH level.In contrast,there showed no clear increase of both H2O2 and NO in non-carcinoma liver cell LO2.CONCLUSION Taken together,a medicinal plant-derived 1,4-napthoquinone,induced iNOS expression by H2O2-dependent JNK activation,caused nitrative stress,finally led to cancer cell death by necroptosis or apoptosis.展开更多
基金supported by Science and Technology Development Fund of Macao Special Administrative Region (078/2016/A2) and Research Fund of University of Macao (MYRG2016-00043-1CMS-QRCM)
文摘OBJECTIVE Glioblastomas(GBM) are the most malignant brain tumors in humans and have a very poor prognosis.New therapeutics are urgently needed.Here,we reported 2-methoxy-6-acetyl-7-methyljuglone(MAM)-induced cell death in U87 and U251 glioma cancer cells.METHODS Cells were cultured and treated with MAM,the cell viability was determined by MTT assay and LDH assay.Intracellular reactive oxygen species(ROS) generation was observed by DCF fluorescence.The protein expression was determined by Western blotting.RESULTS MAM induced glioma cancer cell death without caspase activation.The cell death induced by MAM was attenuated by the pharmacological or genetic blockage of necroptosis signaling,including RIP1 inhibitor necrostatin-1 s(Nec-1 s) and siRNA-mediated gene silencing of RIP1 and RIP3,but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1 H-Indol-3-yl)-3-pentylamino-maleimide(IM54).MAM treated U87 and U251 glioma cancer cells induced RIP1/RIP3 complex formation,ROS level increased,ATP concentration decreased and loss of plasma membrane integrity,further confirmed this process was necroptosis.The essential role of ROS was confirmed by the protective effect of ROS scavenger NAC.Interestingly,MAM induced necroptosis both triggered by RIP1/RIP3 complex and ROS generation.Moreover,MAM induced necroptosis through cytosolic calcium(Ca2 +) accumulation and sustained c-Jun N-terminal kinase(JNK) activation.Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate cell death.Further,we found there exists a feedback loop between RIP1 and JNK activation.Finally,MAM induced necroptosis was inhibited by dicoumarol(a NQO1 inhibitor).Dicoumarol exposed glioma cancer cells were resistant to RIP1/RIP3 complex formation and ROS generation.MAM induced necroptosis was independent of MLKL.CONCLUSION MAM induced non-canonical necroptosis through the NQO1-dependent ROS and RIP1/RIP3 pathway.This study also provided new insights into the molecular regulation of necroptosis in human glioma cancer cells and a promising approach for GBM treatment.
基金The project supported by the Science and Technology Development Fund,Macao S.A.R(FDCT)(021/2012/A1)the Research Fund of University of Macao(MYRG118〔(Y2-L4)-ICMS13-CXP〕
文摘OBJECTIVE To investigate the mechanism of anticancer effect of 2-methoxystypandrone(2-MS),a natural naphthoquinone isolated from Polygonum cuspidatum Sieb.et Zucc.METHODSThree types of cancer cells were investigated in the research(A549,MCF7,B16-F10).Flow cytometer was used to determine ROS/RNS generation.Western blotting was used to detect related protein expression.Apoptosis assay,GSH/GSSG(reduced glutathione/oxidized glutathione)assay were performed using commercial kit.SiRNA knockdown was used to silence cj-un N-terminal kinase(JNK)and iNOS.High-performance liquid chromatography(HPLC)was used to detect the direct reaction of 2-MS with GSH.RESULTS 2-MS induced cytotoxity towards a panel of cancer cells,with less effect on normal cells.2-MS induced necroptosis in A549 cell and apoptosis in B16-F10 and MCF7cells.2-MS increased phosphorylation of JNK in three types of cancer cells.Inhibition of JNK with SP600125 or silencing JNK attenuated 2-MS-induced cell death.JNK also activated iNOS expression and led to nitric oxide(NO)generation in three cancer cells.NO-induced nitrative stress was responsible for DNA damage and necroptosis in A549 cells.NO also inhibited NF-κB expression and induced intrinsic apoptosis in B16-F10 and MCF7cells.Both NO scavenger hemoglobin and silencing iNOS can partially reverse 2-MS-induced cell death.Furthermore,we found that all of these were attributed to induction of hydrogen peroxide(H2O2),which was caused by glutathione(GSH)depletion through interaction of 2-MS with GSH.The interaction was validated through cell-free HPLC analysis.Both the H2O2 scavenger catalase and exogenous GSH can significantly reverse the 2-MS-induced cell death.But catalase did not protect against the decrease in GSH level.In contrast,there showed no clear increase of both H2O2 and NO in non-carcinoma liver cell LO2.CONCLUSION Taken together,a medicinal plant-derived 1,4-napthoquinone,induced iNOS expression by H2O2-dependent JNK activation,caused nitrative stress,finally led to cancer cell death by necroptosis or apoptosis.
