Y320 suppresses non-small cell lung cancer growth by triggering autophagy-dependent pyroptosis through ubiquitin/proteasome-mediated c-FLIP degradation.

Y320, initially identified as an immunomodulator, has been reported to reverse multidrug resistance to chemotherapeutic agents in breast and liver cancers. However, its antitumor activities and underlying mechanisms remain poorly elucidated. In this study, we first investigated the antitumor effects and mechanisms of Y320 in non-small cell lung cancer (NSCLC). Y320 exerted potent antitumor activities against NSCLC both in vitro and in vivo. It induced excessive autophagy, as evidenced by increased autophagosome and autolysosome formation, reduced sequestosome 1 (SQSTM1) protein levels, and conversion of microtubule-associated protein 1 light chain 3 beta (LC3B) from type I to type II. Concurrently, Y320 significantly triggered pyroptosis via the caspase-3/GSDME pathway, as indicated by cellular swelling, membrane blebbing, lactate dehydrogenase (LDH) release, increased propidium iodide (PI) uptake, and elevated levels of cleaved caspase-3 and GSDME-NT. Y320-induced pyroptosis depended on autophagy, and blockade of autophagy at the early stage reversed the occurrence of pyroptosis. Additionally, Y320 downregulated cellular FLICE-like inhibitory protein (c-FLIP) expression through the proteasomal pathway. Importantly, we were the first to verify that knockdown of c-FLIP induced pyroptosis via activation of the caspase-3/GSDME axis. Overexpression of c-FLIP reversed Y320-induced autophagy-dependent pyroptosis. Thus, Y320 induced excessive autophagy-dependent pyroptosis in NSCLC cells by downregulating c-FLIP, ultimately leading to cell death. In conclusion, our study identifies Y320 as a promising therapeutic candidate for NSCLC and elucidates that autophagy-dependent pyroptosis is involved in its antitumor mechanism.