Role And Its Mechanism Of NLRP3/6 In Anisomycin-mediated Antihepatocellular Carcinoma

Background and Purpose: Anisomycin is a small molecule antibiotic derived from bacteria that inhibits peptide chain extension and blocks protein synthesis.The literature and our previous studies have confirmed anisomycin can inhibit the growth of a variety of tumor cells and induce their apoptosis in vitro,but its effects and mechanism on liver tumor biological behaviors are not yet clear.Hepatocellular carcinoma(HCC)is a common malignant tumor characterized by rapid progression,high malignancy and short survival.Since decompensated cirrhosis is usually associated with the occurrence of HCC,there is,at present,a lack of effective,low toxicity and targeted therapies.Inflammation is closely related to the occurrence,development and metastasis of tumors,and inflammasome signals play a vital role in regulating the innate inflammatory immune response and cell death.Therefore,it is of greatly scientific significance to explore the biological mechanism of HCC development and to find effective drug targets.This research attempts to prove the role of NLRP3(NOD-like receptor family pyrin domain containing 3)and NLRP6(NOD-like receptor family pyrin domain containing 6)in anisomycin anti-HCC and its mechanism,so as to provide a new strategy and theoretical support for clinical treatment of HCC.Methods:(1)Anisomycin was used to treat human hepatoma cell Hep3 B,mouse hepatoma cell Hepa1-6 and human hepatocyte LO-2 as a control to detect the influence of anisomycin on hepatoma cell biological behaviors including cell proliferation,apoptosis,adhesion and migration;To determine the role of anisomycin on Wnt and NLRP3 pathways,the expression of proteins in the cell lysate is tested by Western Blot,and the secretion of IL-1β and IL-18 in the cell supernatant is tested by ELISA.To further clarify the relationship between NLRP3 and HCC,the expression of NLRP3 in the liver tumor tissues and adjacent tissues were determined by IHC,Western Blot and qPCR.The direct molecular targets of anisomycin on the cell membrane were determined by the Thermal Drift Detection(TSA)and Surface Plasma Resonance(SPR)of biophysics.The interaction between peroxisome proliferation activated receptors δ(PPARδ),heat shock protein 90(HSP90)and NLRP3 was determined by Co-IP and FRET,and the relationship between upstream and downstream regulation of each molecule was determined by the use of corresponding molecular agonists and inhibitors.Finally,the in vivo effect of anisomycin anti-HCC was detected by the mouse tumor model.The pathological changes of heart,liver,spleen,kidney and the level of ALT,AST,BUN and TNF-α of mice serum were decected 60 days after injection of anisomyin.(2)To prove the role of NLRP6 in anisomycin anti-HCC,the NLRP6 overexpression and knockdown cell lines were constructed via CD513 B and GV298 lentiviral vectors.To further verify the relationship between NLRP3 and NLRP6,the expression differences of NLRP3 and NLRP6 in the normal liver tissue and liver tumor tissue were detected by IHC.Furthermore,NLRP6 and Caspase-8 were regulated to understand the relationship between the NF-κB and NLRP3 signals.Results: Anisomycin could inhibit the proliferation,weaken cell adhesion,inhibit cell migration,and promote apoptosis of hepatoma cells in vitro.TSA and SPR showed that anisomycin acted directly on cell membrane receptor molecule Frizzled 3(FZD3)and FZD6.Anisomycin inhibited the expression of Wnt signaling and up-regulated NLRP3 inflammasome-associated molecules,and promoted the secretion of inflammatory cytokines IL-1β and IL-18.In the liver tumor tissues PPARδ was up-regulated,while NLRP3 was significantly reduced.Co-IP and FRET showed that anisomycin could inhibit the interaction between HSP90 and NLRP3.In vivo tests showed that anisomycin could effectively inhibit the growth of HCC by activating NLRP3,and no obvious damage was seen in the dose of 5.0-12.5 mg/kg.Interestingly,NLRP6 was down-regulated when anisomycin triggered NLRP3.The overexpression of NLRP6 could attenuate the role of anisomycin anti-HCC to a certain extent.Caspase-8 could inhibit NLRP6 and activate NF-κB signal to increase NLRP3.Conclusion: Anisomycin can activate NLRP3 inflammasome activity by FZD6/Wnt/PPARδ/HSP90 signaling pathway,promote apoptosis of hepatoma cells and inhibit the growth of HCC.Anisomycin also inhibits NLRP6 to activate NF-κB by caspase-8,thus inhibiting the indirect negative feedback regulation of NLRP6 on NLRP3.