Cluvenone induces apoptosis via a direct target in mitochondria: a possible mechanism to circumvent chemo-resistance?

摘要

The synthetic caged Garcinia xanthone, cluvenone, has potent and selective cytotoxicity against numerous cancer cell lines including those that are multi-drug resistant. The direct target of this structurally and functionally unique agent is unknown and that of the parent natural product, gambogic acid (GA), presently in clinical trials, is not yet entirely clear. For the first time, using fluorescently labeled GA (GA-Bodipy), we determined that GA-Bodipy localized in mitochondria and was effectively displaced by cluvenone in competition experiments indicating that the direct target of cluvenone resided in mitochondria and was shared by GA. In agreement with these findings, treatment of HeLa cells with cluvenone or GA resulted in disruption of mitochondrial morphology within 4 h. Furthermore, experiments using the potential sensitive JC-1 dye demonstrated that cells treated with 1 μM cluvenone for 1 h had significant loss of MMP compared to control cells. Examination of Cyt c levels in leukemia cells treated with 1 μM cluvenone resulted in a 4-fold increase in levels of both cytosolic and mitochondrial Cyt c. In agreement with Cyt c release, caspase 9 activity was increased 2.6-fold after treatment of cells for 5 h with 1 μM cluvenone. Remarkably, the caspase-9 inhibitor, Z-LEHD-FMK, blocked cluvenone-induced apoptosis in a dose-dependent manner with apoptosis being completely blocked by 10 μM of the inhibitor. In conclusion, cluvenone, an agent with potent cytotoxicity against multi-drug resistant tumor cells, has direct targets in mitochondria thus setting precedence for drug discovery efforts against these targets in the treatment of refractory cancers.