Stigmasterol Induces Cell Apoptosis in Prostate Cancer by Calcium Overload in Mitochondria

Background: Stigmasterol has been reported to inhibit the growth and proliferation of various cancer types, including prostate cancer. However, its inhibitory mechanism in prostate cancer remains unclear. Therefore, this study aimed to investigate the underlying inhibitory mechanisms of stigmasterol in prostate cancer. Methods: Initially, PC-3 and DU145 cells were treated with various concentrations of stigmasterol (0, 1, 2, 5, 10, 20, 50, and 100 mu M). After this, the cell viability of treated cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Moreover, the cell cycle, apoptosis rate, reactive oxygen species (ROS) production, Ca2+ levels in cytosol or mitochondria, and loss of mitochondrial membrane potential (MMP) were determined through functional experiments, including flow cytometry and JC-1 staining. Furthermore, apoptosis-related proteins were evaluated employing Western blot analysis. Additionally, to block mitochondrial Ca2+ influx, the cells were treated with 4, 4 '-diisothiocyanostilbene-2, 2 '-disulfonic acid (DIDS) before stigmasterol treatment. Rescue experiments were conducted to examine the expression of the c-Jun N-terminal kinase-2 (JNK2)/p38 pathway using Western blot analysis. Results: Stigmasterol shortened the G1 phase and expanded the G2/M phase, promoted cell apoptosis, induced ROS production, enhanced loss of MMP, increased Ca2+ overload, and up-regulated apoptosis-related proteins, including cleaved caspase-3, cleaved caspase-9, and cytochrome C, as well as Bcl2-associated X protein (Bax). However, DIDS treatment partially counteracted the afore-mentioned characteristics in prostate cancer cells induced by stigmasterol. Interestingly, DIDS treatment significantly reversed the stigmasterol-induced inhibition of JNK2 and p38 in PC-3 and DU145 cells. Conclusion: Stigmasterol promoted apoptosis in prostate cancer cells through ROS accumulation, loss of MMP, mitochondrial Ca2+ overload, and inhibition of the JNK2/p38 signaling pathway.