Targeting 14-3-3 protein, difopein induces apoptosis of human glioma cells and suppresses tumor growth in mice

14-3-3 protein has emerged as critical regulators of diverse cellular responses. Previous studies found that strong 14-3-3 protein expression was observed and associated with tumor genesis and progression in glioma. Here, we further elucidated the role of 14-3-3 protein in Apoptosis of human glioma U251 and U87 cells by global inhibition of 14-3-3 functions with a general 14-3-3 antagonist, difopein. In vitro, morphological observation and DNA laddering assay showed that difopein-treated glioma cells displayed outstanding Apoptosis characteristics, such as nuclear fragmentation, appearance of membrane-enclosed apoptotic bodies and DNA laddering fragment. Moreover, flow cytometric detection of phosphatidylserine externalization indicated that difopein-induced Apoptosis occurred in a time-dependent manner. Interestingly, inhibiting 14-3-3 with small interfere RNA also induce Apoptosis of human glioma U251 cells. Furthermore, RT-PCR and western blot assay further substantiated that difopein had strong effects to induce glioma cell Apoptosis through down-regulating Bcl-2, up-regulating Bax and activating caspase-9 and Caspase-3. In vivo, retroviral vector was constructed and retroviral-mediated transfer of difopein to glioma was implanted in nude mice. Difopein effectively hindered proliferation and triggered Apoptosis of tumor cells implanted into nude mice. This work not only reveals a critical role of 14-3-3 in Apoptosis suppression in glioma cells, but also identifies and validates 14-3-3 as a potential molecular target for Anticancer therapeutic development.