Targeting 14-3-3 protein, difopein induces apoptosis of human glioma cells and suppresses tumor growth in mice
Targeting 14-3-3 protein, difopein induces apoptosis of human glioma cells and suppresses tumor growth in mice
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DOI：
10.1007/s10495-009-0437-4
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发表时间：
2010-02-01
期刊：
影响因子：
7.2
通讯作者：
Fei, Zhou
中科院分区：
文献类型：
--
作者：
Cao, Weidong;Yang, Xiaoliang;Fei, Zhou
14-3-3蛋白已成为多种细胞反应的关键调节剂。以往的研究发现14-3-3蛋白在胶质瘤中有较强的表达，并与胶质瘤的发生、发展有关。在此，我们进一步阐明了14-3-3蛋白在人胶质瘤U251和U87细胞凋亡中的作用，通过14 - 3 -3拮抗剂difopein全面抑制14-3-3功能。体外形态学观察和DNA梯状条带分析显示，地福平处理的胶质瘤细胞具有明显的凋亡特征，如核碎裂、出现凋亡小体和DNA梯状条带。此外，流式细胞仪检测磷脂酰丝氨酸外化表明，地福平诱导的细胞凋亡发生在一个时间依赖性的方式。有趣的是，用小干扰RNA抑制14-3-3也诱导人胶质瘤U251细胞凋亡。RT-PCR和western blot实验进一步证实，Difopein通过下调Bcl-2、上调Bax、激活caspase-9和caspase-3诱导胶质瘤细胞凋亡。在体内，构建逆转录病毒载体，并将Difopein转移到裸鼠脑胶质瘤中。Difopein有效地抑制了裸鼠移植瘤细胞的增殖，并引发了肿瘤细胞的凋亡。这项工作不仅揭示了14-3-3在胶质瘤细胞凋亡抑制中的关键作用，而且还鉴定和验证了14-3-3作为抗癌治疗开发的潜在分子靶点。
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.