Molecular Mechanisms Of Histon Deacetylase Inhibitor Protects L929Cells From TNFα-Induced Programmed Cell Necrosis

Apoptosis is an active type of cell death, which is also named as programmed celldeath (PCD) as its progress are regulated by series of genes. In the past over years,apoptosis is almost synonymous with programmed cell death, whereas necrosis isdefined as an unregulated and passive cell death; the mechanisms of necrosis areneglected for a long period. The studies in recent years have found that in some celllines, during death receptors-mediated apoptosis, if the activation of caspases areblocked by pan-caspase inhibitor zVAD-fmk, the cells will not undergo apoptosis, butturn to necrosis which is described as Necroptosis. There are no morphologicaldifferences between necroptosis and classical necrosis induced by accident or extremecondition, but more and more studies have proved that necroptosis are regulated bymultiple genes as well, so it is classified as one type of programmed cell death, i.e.,programmed necrosis (hereinafter referred to as necrosis).Many studies have proved that RIPK1/RIPK3kinases are crucial for necrosis.Nrcrostatin-1(Nec-1), a small molecule compounds which target RIP1inhibiting itskinase activity has shown its poteintial to inhibit necrosis both in vitro and in vivo.Necrosis induces the formation of necrosome which contains RIPK1, RIPK3,caspase-8and FADD. Both RIPK1and RIPK3are phosphorylated in the necrosome,and the combination between RIP1and RIP3is also necessary for the complex. Inaddition, the negative regulator of cell necrosis FLIPLcombines with FADD andcaspase-8to form another complex, which protects cells from necrosis in embryonicdevelopment and T lymphocyte activation process. It’s proved recently that RIP3withits downstream protein MLKL and PGAM5forms mitochondria attack complex totransfer necrotic signal to the mitochondria and induce necrosis through mitochondrialdivision proteins Drp-1. Compared with the understanding of apoptosis, we just knowless about how to regulate necroptosis. No doubt, it needs more work to investigateand finally elucidate the molecular mechanism of necrosis.Histone deacetylase (HDAC) inhibitors are the kind of important epigenetic drugs, which become new anti-tumor drugs with characteristic of higher efficiency and lowertoxicity. Among HDAC inhibitor, Vorinostat (SAHA) has been approved by the FADof United States for clinical treatment of cutaneous T-cell lymphoma. More otherselective HDAC inhibitors have entered clinical trials of I or II separately. Themechanisms for HDAC inhibitors include upregulation of histone acetylation in cellsgenome, inducing gene activation by which to mediate the cell cycle arrest, cellgrowth inhibition and/or apoptosis. In addition to anticancer effect, recent studieshave confirmed that HDAC inhibitors also have anti-inflammatory activity. Moreover,HDAC inhibitors can protect cells from cell death in many models, includingtraumatic brain injury, ischemia-reperfusion-induced oxidation damage, heart failureand sepsis-induced liver cell injury, etc. Therefore, the more we know about howHDAC inhibitors works, the more we can do to use these drugs on variety of diseasetreatment.In this study, by using a classic TNFα-induced mouse fibrosarcoma L929cellsnecrosis model, we investigated the effect of HDAC inhibitors on blocking necroticcell death. Our results showed that several HDAC inhibitors, such as SAHA,depsipeptide and MS275all showed the protective effect on necrosis, although withdifferent potential. Further studies were focused on SAHA.We found that even1μMSAHA showed optimal antagonistic effect on necrosis, whereas there were no effectson upregulting of p21expression, arresting cell cycle and inducing apoptosis with thisconcentration. It suggests that the protective effect of SAHA on necrosis isindependent of above known mechanism. We noticed that the protective effect ofSAHA on necrosis needed to pretreated cells for a period of time, it means that SAHAmay change some genes expression by epigenetic regulation. We detected the proteinsexpression of TNFR signaling complexes, and found that SAHA candose-dependently decrease the expression of RIP1, TRAF2, cIAPs, A20and CYLD,but enhanced the expression of FLIPL. Since under the concentration of1μM SAHA,the remarkable change was time-dependent increased FLIPLexpression, suggestingthat SAHA may inhibit the formation of RIP1/FADD/Casp-8Ripoptosome complexvia upregulating the expression of negative regulator FLIPL. The experiments ofknocking down FLIPLexpression and detecting the effect on ripoptosome complex areneeded to be done in the near future.In the detection of TNFα-mediated signaling pathways, by western blottinganalysis, we also found that SAHA could differently regulate these pathways. SAHA enhanced theTNFα-induced activation of NFκB and p38MAPK, but inhibitedthe ROS-induced sustained activation of JNK and intricic activation of Akt. Thesedifferent regulations of SAHA on signal transduction paly improtant roles onmediating antagonistic necrosis which have been confirmed by using specific signalpathway inhibitors seperetely. In addition, we noticed that inhibiting mTOR byrapamycin, a downstream kinase of Akt also showed remarkable effect on antagonisnecrosis, which suggested that initiating autophagy could protectes the cells fromnecrosis in L929cells. Further studies also showed that SAHA lost its protectiveeffect on RIPK1knockdown L929cells, the effect of SAHA on activing NFκB,p38MAPK and inhibiting JNK were decreased. These results indicated that at leastpartially, the protecition effect of SAHA is dependent on regulating RIPK1-mediatedsignal transduction.Conclusion: SAH`A could protect the cells from TNFα-induced necrosis throughupregulating FLIPL,enhancing the activation of p38-NFκB, inhibiting sustainedactivation of JNK and Akt, and initiating autophage.