Abstract 6781: Effect of Qingre Lishi Huoxue prescription on pyroptosis and the expression of related proteins caspase-3/GSDME in esophageal cancer cells

Introduction: Esophageal carcinoma (EC) is one of the most common malignant tumors posing a threat to human's health, with a high disease incidence and the highest death rate in China. This study aims to screen out the Chinese herbs with strong inhibitory effects on the proliferation of four kinds of EC cells EC9706, EC-1, TE-1 and Eca109 from 300 Chinese herbs, and study the effect of Qingre Lishi Huoxue Prescription(QRLSHXP) on the proliferation and on pyroptosis-related proteins such as Caspase-3 and GSDME of four EC cells. Methods: The MTT assay was used to assess the effects of 300 Chinese herbs alcohol extracts on the proliferation of the four esophageal cancer cell lines. Based on efficacy and safety profiles, selected herbs were combined to formulate a prescription. Dose-response relationships were determined. Cell morphology changes were observed under an inverted microscope. DNA damage was assessed using TUNEL staining. Lactate dehydrogenase (LDH) release was measured to evaluate cell membrane integrity. Cell death was analyzed using Annexin V/PI staining and flow cytometry. Western blotting and ELISA were performed to detect the expression levels of pyroptosis-related proteins and cytokines. Results: Eight herbs significantly inhibited all four esophageal cancer cell lines in the initial screening. Toxic herbs were excluded. Four herbs—Sanye Danshen, Jiuguanxue, Shanchagen, and Huaxuedan—were chosen to create the Qingre Lishi Huoxue Prescription (QRLSHXP). This prescription inhibited the proliferation of the cancer cells in a dose- and time-dependent manner.QRLSHXP treatment induced severe cellular swelling, rupture, and content leakage in EC cells. TUNEL staining revealed increased DNA double-strand breaks, indicating enhanced DNA damage. Elevated LDH levels in supernatants suggested compromised membrane integrity. Flow cytometry showed a significant rise in Annexin V/PI double-positive cells, confirming cell death. Western blot analysis indicated increased Cleaved-Caspase-3 and GSDME-N expression, with unchanged total GSDME levels. ELISA results demonstrated significantly higher IL-1β and IL-18 levels in treated groups. Conclusion: QRLSHXP effectively inhibited the proliferation of EC cells and induced pyroptosis, which was likely associated with the activation of the Caspase-3/GSDME pathway. Citation Format: Fuchun Si, Gao Si. Effect of Qingre Lishi Huoxue prescription on pyroptosis and the expression of related proteins caspase-3/GSDME in esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6781.