NQO1 dependent non-canonical necroptosis mediated by ROS and RIP1/RIP3 in parallel in glioma cancer cells

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.

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 M-IT 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-ls （Nec-ls） and siRNA- mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-（1H-Indol-3-yl）-3-pentylamino-maleirnide （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 NQOl-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.