Cucurbitacin I induces immunogenic cell death and synergistically potentiates cisplatin efficacy in epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is characterized by a suppressive tumor immune microenvironment (TIME) that undermines therapeutic efficacy. Immunogenic cell death (ICD) is a promising immunotherapy strategy attributed to ameliorating the suppressive TIME. Cisplatin (CDDP), the first-line chemotherapy for EOC, is hindered by cumulative toxicity. Cucurbitacin I (CuI), a bioactive plant-derived compound, exhibits remarkable antitumor activity in preclinical models. However, its ICD-inducing capability and cisplatin-sensitizing potential in EOC remain uninvestigated.

PURPOSE: This study aims to systematically elucidate the ICD-inducing capability of CuI and its potential to enhance CDDP's antitumor effects in EOC both in vitro and in vivo.

METHOD: The inhibitory effects of CuI on EOC were evaluated at the cellular level. The half-maximal inhibitory concentrations (IC50) of CuI in EOC cell lines were determined via CCK-8 assay. CuI's effects on cell cycle, pyroptosis (Annexin V+/PI+), immune gene expression profile, and GSDME/cleaved-caspase-3 were assessed in ID8/OVCAR8 cells. Pyroptotic pathways were validated using GSDME siRNA and caspase-3 inhibitor (Z-DEVD-FMK). Following the Nomenclature Committee on Cell Death (NCCD) guidelines for ICD inducer validation, we systematically assessed damage-associated molecular patterns (DAMPs) release (CALR, ERp57, HMGB1, HSPs), dendritic cell (DC) maturation (flow cytometry analysis of murine bone marrow-derived dendritic cells (BMDCs) co-cultured with CuI-pretreated ID8 cells), and in vivo tumor vaccine potency. Intraperitoneal xenograft models evaluated CuI's antitumor efficacy and immune infiltration. Finally, CuI-CDDP synergy was tested in EOC cell lines and immunocompetent models; targets were predicted by bioinformatics (Connectivity Map and molecular docking) and validated by Surface Plasmon Resonance (SPR) to elucidate mechanistic interactions.

RESULT: CuI induced caspase-3/GSDME-mediated pyroptosis in EOC cells, driving the release of ICD-related DAMPs, including surface-exposed CRT/ERp57, ATP, and HMGB1. Consequently, CuI-treated EOC cells promoted DC maturation, significantly increasing the proportion of CD80+CD86+ DCs in tumor-draining lymph nodes (from 16.07±1.02 % to 23.47±1.94 %). In ID8-luc intraperitoneal xenograft, CuI could effectively suppress ascites formation by 41.4 % and was associated with an approximately 2.8-fold increase in CD8+T cell infiltration. Furthermore, the combination of CuI and CDDP synergistically enhanced pyroptosis in vitro. This synergy translated to superior antitumor effects in the ID8 xenograft model, achieving a tumor growth inhibition (TGI) of 66.02 %. This potent efficacy was accompanied by enhanced CD8+T cell infiltration, a reduction in PD-1 expression on CD8+T cells, and an approximately 1.78-fold increase in the splenic effector memory CD8+T cell (CD44+CD62L-) population compared to the control. Mechanistically, CuI may enhance CDDP-induced intracellular DNA damage and reactive oxygen species accumulation by inhibiting EGFR activation.

CONCLUSION: CuI is a novel ICD inducer that elicits caspase-3/GSDME-mediated pyroptosis and boosts antitumor immunity in EOC. Notably, it enhances the chemosensitivity of EOC to CDDP, offering a promising strategy for EOC treatment.