2-Methoxystypandrone induces iNOS expression by H2O2-dependent JNK activation for multiple cancer cell death

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.