Molecular Mechanisms Of Necroptosis Induced By Neoalbaconol In Cancer Cells

Abstract:Programmed cell death (PCD) plays a fundamental role in development and tissue homeostasis.There are three major forms of programmed cell death in mammalian cells:apoptosis, autophagic cell death, and regulated necrosis, sometimes referred to as necroptosis. In2005, researchers introduced the neologism necroptosis to describe one instance of regulated (as opposed to accidental) necrotic cell death.Regulated necrotic cells exhibit some common morphological features, including an increasingly translucent cytoplasm, swelling of organelles, minor ultrastructural modifications of the nucleus (specifically, dilatation of the nuclear membrane and condensation of chromatin into small, irregular, circumscribed patches) and increased cell volume, culminating in the disruption of the plasma membrane. It should be noted that necroptosis is RIPK3/RIPK1-dependent, but caspase-independent. Different from apoptosis, necroptosis can elicit immuno-response; participate in viral infection, ischemic injuries, and neurodegenerative disease.In a previous study, our group reported that neoalbaconol (NA), a novel small-molecular compound isolated from the fungus, Albatrellus confluens, could cause necroptotic cell death in narsopharygeal carcinoma cells. Here, we found that necroptosis was markedly induced in different cancer cell lines, which was confirmed by the presence of necrotic morphology, rescued by the necroptosis inhibitor necrostatin-1(Nec-1) and knockdown RIPK1/RIPK3by siRNA.To elucidate the molecular mechanism of neoalbaconol-induced necroptosis, firstly, we observed the ubiquitination of RIPK1. Neoalbaconol abolished the RIPK1ubiquitination by down-regulating the E3ubiquitin ligases, c-IAP1/2and TRAF2. The RIPK1de-ubiquitination and NIK accumulation activated the non-canonical NF-κB pathway, stimulating the transcRIPKtion of TNFα. Neoalbaconol reduced the phosphorylation and nuclear translocation of p65to inhibit the canonical NF-κB pathway. Specifically, we showed that neoalbaconol treatment altered the functional state of the TNFα from pro-survival through the canonical NF-κB pathway to promote cell death mediated by the RIPK/non-canonical NF-κB pathway.To clarify the relationship between necroptosis induced by neoalbaconol and mitochondra, we performed the antioxidant NAC in neoalbaconol-treated cells. We found that neoalbaconol caused RIPK3-mediated ROS production, which contributed to cell death. Also, neoalbaconol inhibited mitochondrial respiration, and reduced the production of intracellular ATP, that supports the idea that energy metabolism affects cell-death mechanisms.In summary, our study provides a mechanistic explanation for the observed ability of neoalbaconol to stimulate cancer cell death and defines a novel biological role for REPK1/NF-κB in the regulation of cellular signaling. To further examine whether neoalbaconol or other small-molecular natural compounds possess similar effects on other cell types, especially in human cancer cells, will be interesting to examine in the future.