Investigation On The Regulatory Molecular Mechanism Of Pyroptosis And Apoptosis In Hepatocellular Carcinoma Cells By Xanthoangelol From Angelica Keiskei Koidzumi

Background : Xanthoangelol(XAG),one of the major components of chalcone compounds in Angelica keiskei Koidzumi,is widely used in functional supplements or drug research development due to its high pharmacological activity and low toxicity.Pyroptosis has recently been described as a novel mode of programmed cell death limiting the malignant progression of tumors.Correlative studies have demonstrated that XAG significantly inhibits the proliferation of hepatocellular carcinoma(HCC)cells,whereas its specific effects on caspase-1-mediated inflammatory pathway in HCC have not been sufficiently explored.The purpose of this project is to evaluate cytotoxic effects of XAG-regulated caspase-1-dependent signaling pathway on HCC and to discuss the corresponding potential mechanisms.The involvement of NF-κB,mitogen-activated protein kinases(MAPKs)signaling pathway in XAG-induced anti-hepatocellular carcinoma effects will also be explored.Methods:Exploration of the inhibitory effect of XAG on Hu H7 and Hep G2 by CCK-8assay,Annexin V-FITC/PI double-staining and PI staining.Then,we explored the function of pyroptosis in XAG against HCC cells.Observation of morphological properties of pyroptotic cells by optical microscopy;detection of intracellular DNA damage by TUNEL staining assay;determination of GSDMD and GSDMD-N protein expression by Western Blot,supplemented by LDH release assay to verify the cell membrane integrity.Mechanistically,the caspase-1-mediated pyroptosis pathway was evaluated for XAG anti-tumor effects.q RT-PCR and Western Blot assays were conducted at both m RNA and protein levels to detect pyroptosis-related proteins expression,as well as ELISA assay to verify IL-18 and IL-1β levels in cell supernatants.Inhibition of pyroptosis pathway using MCC950(NLRP3 inhibitor)and VX-765(caspase-1 specific inhibitor)to observe effectiveness of XAG on HCC cells.Western Blot assay was used to detect proteins expression for verification of the implications of NF-κB and MAPKs signaling pathways on XAG anti-tumor effects.Confirmation of whether caspase-1expression mediates the XAG-induced apoptotic pathway.In Hep G2 cells,the apoptotic morphology was monitored by light microscopy;the proportion of apoptotic cells was examined by flow cytometric assay;the expression of apoptotic proteins and caspase-1was detected by Western Blot assay.To conclude,Western Blot and flow cytometric assays were performed to verify the effect of VX-765 on XAG-induced apoptosis..Main results: It was found that XAG significantly reduced HCC cell viability and promoted apoptosis in a dose-dependent manner.And after XAG treatment,cell cycles of Hu H7 and Hep G2 were blocked in G1/S phase and G2/M phase,respectively.Initially,it was shown that XAG in HCC cell lines Hu H7 and Hep G2 may inspire two different death approaches for inhibition of cell proliferation.Microscopically,we observed that XAG induced Hu H7 cell membrane swelling and even rupture.GSDMD-N acts as a key perforation factor of the pyroptosis pathway,binding to and multimerizing with phospholipids on the membrane to form pores.Western Blot and LDH release assays revealed a significant increase of GSDMD-N expression and LDH levels in the supernatant,indicating that XAG may activate pyroptosis in Hu H7 cells.We further validated pyroptotic proteins expression both at the transcriptional as well as the translational level and observed that XAG upregulated NLRP3,Pro-caspase-1,Cleaved-caspase-1 and promoted the release of secreted proteins IL-18 and IL-1β.When XAG combined with inhibitors(MCC950,VX-765),the perforating ability of XAG on HCC cells was diminished,and the expression of pyroptotic proteins and the secretion of IL-18 and IL-1β in the supernatant were also significantly decreased.The above results suggest that XAG exhibits cytotoxic effects through induction of pyroptosis in Hu H7 cells by activating the NLRP3/caspase-1/GSDMD pathway;NLRP3 inflammatory is involved in pyroptosis in the process of XAG inhibition of HCC.Additionally,XAG downregulated proteins expression of NF-κB and MAPKs signaling pathways to prohibit liver cancer cell proliferation.In contrast,there was no notable phenomenon of pyroptosis,but instead,the distinct apoptotic morphology was found accompanied by caspase-1activation in Hep G2 cells.The effect of caspase-1 expression on XAG-induced apoptosis was further verified.Flow cytometric assays as well as Western Blot declared that VX-765 attenuated XAG-induced apoptosis in Hep G2 cells with low GSDMD expression.Conclusions and implications : XAG inhibits HCC cell growth by inducing caspase-1-dependent pyroptotic and apoptotic cell death(GSDMD-low cell types).XAG also exerts anti-tumor effects by inhibiting the activation of NF-κB and MAPKs signaling pathways.Importantly,this study initially confirmed the correlation between liver cancer and caspase-1 and the mechanism of anti-liver cancer of XAG,providing evidence for chalcones targeting caspase-1 protein to regulate liver cancer cell death.