Gambogic acid induces GSDME dependent pyroptotic signaling pathway via ROS/P53/Mitochondria/Caspase-3 in ovarian cancer cells

Abstract

Gambogic acid (GA) is a naturally active compound extracted from the Garcinia hanburyi with various anticancer activities. However, whether GA induces pyroptosis (a newly discovered inflammation-mediated programmed cell death mechanism) in ovarian cancer (OC) has not yet been reported. This study revealed that GA treatment reduced cell viability by inducing pyroptosis in OC cell lines. Typical pyroptosis morphological manifestations such as cell swelling with large bubbles and loss of cell membrane integrity, were observed. Cleaved caspase-3 and GSDME-N levels increased after GA treatment, and knocking out GSDME or using a caspase-3 inhibitor could switch GA-induced cell death from pyroptosis to apoptosis, indicating GA induced caspase-3/GSDME-dependent pyroptosis. Furthermore, this research indicated that GA significantly increased reactive oxygen species (ROS) and p53 phosphorylation. OC cells pretreated with ROS inhibitor N-Acetylcysteine (NAC) and the specific p53 inhibitor pifithrin-μ could completely reverse the pyroptosis post-treatment. Elevated p53 and phosphorylated p53 reduced mitochondrial membrane potential (MMP) and Bcl-2, increase the expression of Bax, and damage mitochondria by releasing cytochrome c to activate the downstream pyroptosis pathway. Different doses of GA inhibited tumor growth in ID8 tumor-bearing mice, and high-dose GA increased in tumor-infiltrating lymphocytes CD3, CD4, and CD8 were detected in tumor tissues. Notably, the expressions of GSDME-N, cleaved caspase-3 and other proteins were increased in tumor tissues with high-dose GA groups. These findings demonstrate that GA-treated OC cells could induce GSDME-mediated pyroptosis through the ROS/p53/mitochondria signaling pathway and caspase-3/-9 activation. Thus, GA is a promising therapeutic agent for OC treatment.