Zang Y, Beard R L, Chandraratna R A, Kang J X
Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Cell Death Differ. 2001 May;8(5):477-85. doi: 10.1038/sj.cdd.4400843.
The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of apoptosis in a variety of tumor cell types. However, the mechanism of its action remains to be elucidated. Recent studies suggest that the lysosomal protease cathepsin D, when released from lysosomes to the cytosol, can initiate apoptosis. In this study, we examined whether cathepsin D and free radicals are involved in the CD437-induced apoptosis. Exposure of human leukemia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicated by caspase activation, phosphatidylserine exposure, mitochondrial alterations and morphological changes. Addition of the antioxidants alpha-tocopherol acetate effectively inhibited the CD437-induced apoptosis. Measurement of the intracellular free radicals indicated a rise in oxidative stress in CD437-treated cells, which could be attenuated by alpha-tocopherol acetate. Interestingly, pretreatment of cells with the cathepsin D inhibitor pepstatin A blocked the CD437-induced free radical formation and apoptotic effects, suggesting the involvement of cathepsin D. However, Western blotting revealed no difference in cellular quantity of any forms of cathepsin D between control cells and CD437-treated cells, whereas immunofluorescence analysis of the intracellular distribution of cathepsin D showed release of the enzyme from lysosomes to the cytosol. Labeling of lysosomes with lysosomotropic probes confirmed that CD437 could induce lysosomal leakage. The CD437-induced relocation of cathepsin D could not be prevented by alpha-tocopherol acetate, suggesting that the lysosomal leakage precedes free radical formation. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to block these effects of CD437, suggesting that the action of CD437 is RAR-independent. Taken together, these data suggest a novel lysosomal pathway for CD437-induced apoptosis, in which lysosomes are the primary target and cathepsin D and free radicals act as death mediators.
新型合成类视黄醇6-[3-(1-金刚烷基)-4-羟基苯基]-2-萘甲酸（AHPN/CD437）已被证明是多种肿瘤细胞类型中一种有效的凋亡诱导剂。然而，其作用机制仍有待阐明。最近的研究表明，溶酶体蛋白酶组织蛋白酶D从溶酶体释放到细胞质中时可引发凋亡。在本研究中，我们检测了组织蛋白酶D和自由基是否参与CD437诱导的凋亡。将人白血病HL-60细胞暴露于CD437导致迅速诱导凋亡，这通过半胱天冬酶激活、磷脂酰丝氨酸暴露、线粒体改变和形态变化得以表明。添加抗氧化剂醋酸α-生育酚可有效抑制CD437诱导的凋亡。细胞内自由基的测量表明，经CD437处理的细胞中氧化应激增加，而醋酸α-生育酚可使其减弱。有趣的是，用组织蛋白酶D抑制剂胃蛋白酶抑制剂A预处理细胞可阻断CD437诱导的自由基形成和凋亡效应，表明组织蛋白酶D参与其中。然而，蛋白质免疫印迹法显示对照细胞和经CD437处理的细胞之间任何形式的组织蛋白酶D的细胞数量没有差异，而组织蛋白酶D细胞内分布的免疫荧光分析显示该酶从溶酶体释放到细胞质中。用溶酶体亲和探针标记溶酶体证实CD437可诱导溶酶体渗漏。醋酸α-生育酚不能阻止CD437诱导的组织蛋白酶D重新定位，表明溶酶体渗漏先于自由基形成。此外，视黄酸核受体（RAR）拮抗剂未能阻断CD437的这些效应，表明CD437的作用不依赖于RAR。综上所述，这些数据提示了一种CD437诱导凋亡的新型溶酶体途径，其中溶酶体是主要靶点，组织蛋白酶D和自由基作为死亡介质发挥作用。
