Necroptosis is a form of programmed
necrosis mediated by signaling complexes with receptor-interacting
protein 1 (RIP1) and RIP3
kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in
2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29
colon cancer cells. With this formation, mitochondrial
reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and
ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) 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-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered 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 necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and
ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of
manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic
calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human
colon cancer cells.