Ge Xiaokang, Fu Ya-Min, Li Yi-Qi, Meadows Gary G
Cancer Prevention and Research Center, Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164-6510, USA.
Arch Biochem Biophys. 2002 Jul 1;403(1):50-8. doi: 10.1016/S0003-9861(02)00211-4.
Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.
酪氨酸（Tyr）和苯丙氨酸（Phe）的缺失会抑制人A375黑色素瘤细胞的生长并诱导程序性细胞死亡（凋亡）。在此，我们发现该过程需要半胱天冬酶的激活和线粒体细胞色素c的释放。在含有10%透析胎牛血清的无Tyr/Phe培养基中培养A375细胞，会导致类似半胱天冬酶-3活性的激活。这伴随着细胞活力下降和凋亡增加。Tyr/Phe缺失还会刺激DNA修复酶聚（ADP-核糖）聚合酶（PARP）的蛋白水解切割。蛋白质印迹分析表明，半胱天冬酶3、7、8和9会因Tyr/Phe的缺失而被激活。Tyr/Phe缺失会降低线粒体膜电位，诱导Bid的切割，增加Bax从细胞质向线粒体的转位，并导致细胞色素c从线粒体释放到细胞质中。广谱细胞可渗透的半胱天冬酶抑制剂苄氧羰基-Val-Ala-Asp-氟甲基酮（Z.VAD.fmk）几乎完全抑制了因Tyr/Phe缺失引起的凋亡。该抑制剂抑制Bid的切割、细胞色素c从线粒体释放到细胞质以及PARP的切割。癸基泛醌，一种线粒体通透性转换孔抑制剂，不抑制半胱天冬酶8的激活，但抑制细胞色素c的释放、半胱天冬酶9的激活和凋亡的诱导。这些结果表明，半胱天冬酶的激活、Bid的切割以及细胞色素c从线粒体的释放是Tyr/Phe缺失诱导凋亡所必需的。
