Abstract
BackgroundPyroptosis belongs to a novel inflammatory programmed cell death pathway, with the possible prognosis of endometrial cancer related to the terminal protein GSDMD. Hydrogen exerts a biphasic effect on cancer by promoting tumor cell death and protecting normal cells, which might initiate GSDMD pathway-mediated pyroptosis.MethodsWe performed immunohistochemical staining and western immunoblotting analysis to observe expression of NLRP3, caspase-1, and GSDMD in human and xenograft mice endometrial cancer tissue and cell lines. We investigated treatment with hydrogen could boost ROS accumulation in endometrial cancer cells by intracellular and mitochondrial sources. GSDMD shRNA lentivirus was used to transfect endometrial cancer cells to investigate the function of GSDMD protein in pyroptosis. Propidium iodide (PI) staining, TUNEL assay, measurement of lactate dehydrogenase (LDH) release and IL-1β ELISA were used to analysis pyroptosis between hydrogen-supplemented or normal culture medium. We conducted in vivo human endometrial tumor xenograft mice model to observe anti-tumor effect in hydrogen supplementation.ResultsWe observed overexpression of NLRP3, caspase-1, and GSDMD in human endometrial cancer and cell lines by IHC and western immunoblotting. Hydrogen pretreatment upregulated ROS and the expression of pyroptosis-related proteins, and increased the number of PI- and TUNEL-positive cells, as well as the release of LDH and IL-1β, however, GSDMD depletion reduced their release. We further demonstrated that hydrogen supplementation in mice was sufficient for the anti-tumor effect to inhibit xenograft volume and weight of endometrial tumors, as mice subjected to hydrogen-rich water displayed decreased radiance. Tumor tissue sections in the HRW groups presented moderate-to-strong positive expression of NLRP3, caspase-1 and GSDMD. Hydrogen attenuated tumor volume and weight in a xenograft mouse model though the pyroptotic pathway.ConclusionsThis study extended our original analysis of the ability of hydrogen to stimulate NLRP3 inflammasome/GSDMD activation in pyroptosis and revealed possible mechanism (s) for improvement of anti-tumor effects in the clinical management of endometrial cancer.
Highlights
Pyroptosis belongs to a novel inflammatory programmed cell death pathway, with the possible prognosis of endometrial cancer related to the terminal protein gasdermin D (GSDMD)
Pyroptosis-related protein NLRP3, caspase-1, and GSDMD are overexpressed in endometrial cancer tissue Upregulated expression of NLRP3, caspase-1, and GSDMD were detected in endometrial cancer tissues (N = 546) compared with normal endometrium (N = 35) in TCGA database, significantly difference was seen in NLRP3 and GSDMD in sample type and histological types (P < 0.05)
Based on the TCGA data above, in order to make a foundation for the follow-up hydrogen related mechanical pyroptotic experiment, we aimed to explore whether pyroptotic phenomena exist in endometrial cancer
Summary
Pyroptosis belongs to a novel inflammatory programmed cell death pathway, with the possible prognosis of endometrial cancer related to the terminal protein GSDMD. Hydrogen exerts a biphasic effect on cancer by promoting tumor cell death and protecting normal cells, which might initiate GSDMD pathway-mediated pyroptosis. Endometrial cancer is the most common gynecologic cancer. The treatment of patients without fertility requirements is still based on surgery, and those who possess high risk factors require radiotherapy or chemotherapy that kills cancer cells by augmenting reactive oxygen species (ROS)-mediated stress and activating apoptosis [2]. Since patients with endometrial cancer have elevated oxidative stress and systemic inflammatory conditions caused by metabolic syndrome, we hypothesized that inflammatory pathways were important as estrogen metabolism aspects of endometrial cancer, and that activation of the inflammationmediated cell death pathway may facilitate the elimination of tumor cells
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