Alpinumisoflavone triggers GSDME-dependent pyroptosis in esophageal squamous cell carcinomas.

Abstract

Esophageal squamous cell carcinoma (ESCC) presents a common human malignancy in the digestive system. We aimed to explore the critical effects of alpinumisoflavone (AIF) on ESCC in vitro and in vivo. The cell counting kit-8 assay was used to determine cell viability. Colony formation assay was employed to examine the effect of AIF on the long-term growth of ESCC cells. Cell apoptosis was determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Cell morphologies were observed by light microscopy. The enzyme-linked immunosorbent assay was performed to examine the lactate dehydrogenase release from AIF-treated cells. Immunofluorescent labeling was utilized to examine AIF-induced GSDME expression. Western blot was employed to determine the expression levels of the associated proteins. Immunohistochemistry was performed to determine the localization and expression of the associated proteins in mice tumor tissues. AIF inhibited ESCC cell viability and suppressed cell growth in a dose- and time-dependent fashion. Results showed that AIF promoted apoptosis in ESCC cells. Meanwhile, our results also showed that AIF triggered pyroptotic cell death in ESCC, which was mediated by gasdermin E (GSDME) cleavage. In addition, our experiments provided experimental evidence that AIF-induced GSDME cleavage was dependent on caspase-3 activation. Moreover, the inhibition of GSDSE by knockdown was able to switch the form of cell death from pyroptosis to apoptosis. Furthermore, the results from the xenograft animal model also supported our findings in vitro that AIF was able to promote GSDME-mediated pyroptotic cell death in ESCC. AIF inhibited ESCC growth in vitro and in vivo by triggering GSDME-mediated pyroptotic cell death, which is dependent on caspase-3 activation.