Muscarinic acetylcholine M1 receptor regulates pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway

Abstract

Objective To determine the effect of muscarinic acetylcholine M1 receptor (CHRM1) on pyroptosis of prostate cancer cells and investigate its possible molecular mechanism. Methods Pirenzepine (PIN), a specific CHRM1 antagonist, and bethanechol (BETH), its agonist, were used to treat PC-3 cells at different doses for different time periods (12, 24 and 48 h). Then a stable PC-3 cell strain cells with CHRM1 knockdown was established by lentivirus infection. A knockdown cell model of gasdermin D (GSDMD) was constructed by plasmid transfection. Cell proliferation ability was detected by CCK8 assay, LDH release level was measured by lactate dehydrogenase (LDH) cytotoxicity assay, and the rate of PI positive cells after staining with Hoechst-PI and Annexin V-PI was observed by flow cytometry and fluorescence inversion microscopy. Cell pyroptosis was observed by transmission electron microscopy and protein expression levels related to pyroptosis pathway were detected by Western blotting. Results The specific inhibitor of CHRM1, pirenzepine, and lentivirus knockdown of CHRM1 reduced the cell viability of PC-3 cells (P < 0.05), and increased the rate of PI positive cells significantly (P < 0.01). At the same time, the phenomenon of cell pyroptosis was observed under electron microscope, and the treatment of the BETH enhanced the cell viability (P < 0.05) and reduced the rate of PI positive cells in a certain concentration range (P < 0.01). After the down-regulation of CHRM1 by drugs and lentiviruses, the proteins levels of Caspase-1/GSDMD pathway, Caspase-1, cleaved Caspase-1, apoptosis associated speck-like protein (ASC), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and GSDMD were significantly increased (P < 0.05). After the GSDMD was knocked down, the rate of PI positive cells was decreased (P < 0.05), LDH release was reduced (P < 0.001), and the levels of related proteins were reduced significantly (P < 0.01). Treatment of CHRM1 inhibitor or knockdown of CHRM1 had no effects on the rate of PI positive cells, LDH release, and the levels of related proteins. Conclusion Down-regulation of CHRM1 induces pyroptosis of prostate cancer cells through Caspase-1/GSDMD pathway.

Keywords

• cell pyroptosis
• muscarinic acetylcholine m1 receptor
• prostate cancer
• caspase-1
• gsdmd