Paris saponin VII induces Caspase-3/GSDME-dependent pyroptosis in pancreatic ductal adenocarcinoma cells by activating ROS/Bax signaling

Objective: Paridis Rhizoma (Chonglou in Chinese), a traditional Chinese herbal medicine, has been shown have strong anti-tumor effects. Paris saponin VII (PSVII), an active constituent isolated from Paridis Rhizoma, was demonstrated to significantly suppress the proliferation of BxPC-3 cells in our previous study. Here, we aimed to elucidate the anti-pancreatic ductal adenocarcinoma (PDAC) effect of PSVII and the underlying mechanism.

Methods: Cell viability was determined by CCK-8, colony formation, and cell migration assays. Cell Apoptosis and Reactive Oxygen Species (ROS) production were measured by flow cytometry with annexin V/propidine iodide (Annexin V/PI) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), respectively. Pyroptosis was evaluated by morphological features, Hoechst 33342/PI staining assay, and release of Lactate Dehydrogenase (LDH). JC-1 Fluorescent Dye was employed to measure mitochondrial membrane potential. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to determine the levels of proteins or mRNAs. The effect in vivo was assessed by a xenograft tumor model.

Results: PSVII inhibited the viability of PDAC cells (BxPC-3, PANC-1, and Capan-2 cells) and induced gasdermin E (GSDME) cleavage, as well as the simultaneous cleavage of Caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). Knockdown of GSDME shifted PSVII-induced Pyroptosis to Apoptosis. Additionally, the effect of PSVII was significantly attenuated by Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK), on the induction of GSDME-dependent Pyroptosis. PSVII also elevated intracellular ROS accumulation and stimulated Bax and Caspase-3/GSDME to conduct Pyroptosis in PDAC cells. The ROS scavenger N-acetyl cysteine (NAC) suppressed the release of LDH and inhibited Caspase-9, Caspase-3, and GSDME cleavage in PDAC cells, ultimately reversing PSVII-induced Pyroptosis. Furthermore, in a xenograft tumor model, PSVII markedly suppressed the growth of PDAC tumors and induced Pyroptosis.

Conclusion: These results demonstrated that PSVII exerts therapeutic effects through Caspase-3/GSDME-dependent Pyroptosis and may constitute a novel strategy for preventing chemotherapeutic resistance in patients with PDAC in the future.

Caspase-3; gasdermin E; pancreatic ductal adenocarcinoma; paris saponin VII; pyroptosis; reactive oxygen species.