Apoptosis induced by genipin in human leukemia K562 cells: Involvement of c-Jun N-terminal kinase in G2/M arrest

Abstract

Aim:To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.Methods:The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis.Results:Genipin reduced the viability of K562 cells with an IC50 value of approximately 250 mol/L. Genipin 200-400 mol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 mol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G2/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 mol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.Conclusion:These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand. © 2011 CPS and SIMM All rights reserved.