Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells

Aim: Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy. Methods: The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg.kg(1).d(1)) was intraperitoneally injected to BEL-7402-bearing mice for 21 days. Results: Platycodin D (5-40 mu mol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70 +/- 3.99, 24.30 +/- 2.30 and 19.70 +/- 2.36 mu mol/L at 24, 48 and 72 h, respectively. Platycodin D (5-20 mu mol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5-20 mu mol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 mu mol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 mu mol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg.kg(1).d(1)) significantly reduced relative tumor volume with decreased body weight. Conclusion: Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors