Protein phosphatase 2A mediates JS‐K‐induced apoptosis by affecting Bcl‐2 family proteins in human hepatocellular carcinoma HepG2 cells

摘要

Abstract Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS‐K‐induced apoptosis by affecting Bcl‐2 family protein. JS‐K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS‐K caused a dose‐ and time‐dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS‐K‐ induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase‐9/3. Moreover, JS‐K‐treatment could lead to the activation of protein phosphatase 2A‐C (PP2A‐C), decrease of anti‐apoptotic Bcl‐2 family‐protein expression including p‐Bcl‐2 (Ser70), Bcl‐2, Bcl‐xL, and Mcl‐1 as well as the increase of pro‐apoptosis Bcl‐2 family‐protein including Bim, Bad, Bax, and Bak. Furthermore, JS‐K caused a marked increase of intracellular NO levels while pre‐treatment with Carboxy‐PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre‐treatment with Carboxy‐PTIO attenuated the JS‐K‐induced up‐regulation of PP2A, Cyt c, and cleaved‐caspase‐9/3 activation. The silencing PP2A‐C by siRNA could abolish the activation of PP2A‐C, down‐regulation of anti‐apoptotic Bcl‐2 family‐protein (p‐Bcl‐2, Bcl‐2, Bcl‐xL, and Mcl‐1), increase of pro‐apoptosis Bcl‐2 family‐protein (Bim, Bad, Bax, and Bak) and apoptotic‐related protein (Cyt c, cleaved caspase‐9/3) that were caused by JS‐K in HepG2 cells. In addition, pre‐treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS‐K. In summary, NO release from JS‐K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl‐2 family proteins.