ABSTRACT
Objective: To investigate the effect of platycodin D on necroptosis of prostate cancer PC-3 cells, and to explored its mechanism related to forkhead transcription factor O3a (FOXO3a). Methods: PC-3 cells were treated with caspase inhibitor Z-VAD-FMK or caspase-3-specific inhibitor AC-DEVD-CHO, and then were exposed to different concentrations of platycodin D for 72 h. The survival rate of PC-3 cells was detected by MTT method. The cell morphology and membrane integrity of PC-3 cells treated with platycodin D were detected by typan blue staining and lactic dehydrogenase (LDH) release test, respectively. The expression levels of key necroptosis factors including mixed lineage kinase domain-like (MLKL), phospho-MLKL (p-MLKL) and its tetramer, as well as FOXO3a and its downstream molecules including factor-associated suicide ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proteins in PC-3 cells treated with different concentrations of platycodin D were detected by Western blotting. The expression level of FOXO3a protein in the nucleus of PC-3 cells was detected by immunofluorescence. After PC-3 cells were transfected with FOXO3a-siRNA or the negative control (NC)-siRNA by liposome, and the expression levels of FOXO3a, FasL and TRAIL proteins were detected by Western blotting. The cell viability and cell membrane integrity of PC-3 cells after FOXO3a gene silencing and treatment with platycodin D were detected by MTT method, typan blue staining and LDH release test, respectively. Results: The survival rate of PC-3 cells treated with platycodin D was decreased in a concentration-dependent manner. There was no significant difference in the survival rate of PC-3 cells treated with DMSO, Z-VAD-FMK and AC-DEVD-CHO combined with the same concentration of platycodin D (all P > 0.05). After the treatment with platycodin D, the trypan blue staining rate and LDH release rate of PC-3 cells were significantly increased (both P < 0.05). The expression levels of MLKL, p-MLKL and its tetramer in PC-3 cells treated with platycodin D were up-regulated (all P < 0.05), and the effect on p-MLKL tetramer was the most significant. Platycodin D also promoted the transposition of FOXO3a into the nucleus, and up-regulated the expressions of FOXO3a and its downstream molecules FasL and TRAIL (all P < 0.05). After transfection with FOXO3a-siRNA, the expression level of FOXO3a protein in PC-3 cells was down-regulated (P < 0.05). Compared with NC-siRNA + platycodin D group, the survival rate of PC-3 cells in FOXO3a-siRNA + platycodin D group was increased (P < 0.05), and the positive rate of typan blue staining and LDH release rate were decreased (both P < 0.05). Conclusion: Platycodin D can induce caspase-independent necroptosis of prostate cancer PC-3 cells, which is involved in FOXO3a pathway and promoting the phosphorylation of MLKL.