Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis

Pyroptosis, a type of programmed cell death (PCD), is characterized by cell swelling with bubbles, and the release of inflammatory cell cytokines. Cucurbitacin B (CuB), extracted from muskmelon pedicel, is a natural bioactive product that could effectively exert anti-tumor activities in lung cancer. However, the exact molecular mechanisms and the direct targets of CuB in non-small cell lung cancer (NSCLC) remain to be discovered. Here, we firstly found that CuB exerted an anti-tumor effect via pyroptosis in NSCLC cells and NSCLC mice models. Next, based on the molecular docking and cellular thermal shift assay (CETSA), we identified that CuB directly bound to Toll-like receptor 4 (TLR4) to activate the NLRP3 inflammasome, which further caused the separation of N- and C-terminals of Gasdermin D (GSDMD) to execute pyroptosis. Moreover, CuB enhanced the mitochondrial reactive oxygen species (ROS), mitochondrial membrane protein Tom20 accumulation, and cytosolic calcium (Ca2+) release, leading to pyroptosis in NSCLC cells. Silencing of TLR4 inhibited CuB-induced pyroptosis and decreased the level of ROS and Ca2+ in A549 cells. In vivo study showed that CuB treatment suppressed lung tumor growth in mice via pyroptosis without dose-dependent manner, and CuB at 0.75 mg/kg had a better anti-tumor effect compared to the Gefitinib group. Taken together, our findings revealed the mechanisms and targets of CuB triggering pyroptosis in NSCLC, thus supporting the notion of developing CuB as a promising therapeutic agent for NSCLC. Cucurbitacin B (CuB) could induce pyroptosis in NSCLC via promoting NLRP3 inflammasome activation in vivo and in vitro. It directly bound to TLR4 to activate NEK7/NLRP3 inflammasome, promote mitochondrial ROS generation, Tom20 accumulation, and Ca2+ aggregation. Moreover, silencing TLR4 inhibited NLRP3 inflammasome activation, and the level of ROS and Ca2+ were decreased, thereby, inhibiting CuB-induced pyroptosis in NSCLC cells. In vivo study showed that CuB suppressed tumor growth through stimulating pyroptosis in NSCLC. Collectively, this study revealed that CuB could trigger TLR4/NLRP3/GSDMD-dependent pyroptosis via up-regulating ROS and Ca2+ levels to suppress NSCLC. [Display omitted]