Lock Edward A, Reed Celia J, Kinsey Gilbert R, Schnellmann Rick G
Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.
Toxicology. 2007 Jan 5;229(1-2):79-90. doi: 10.1016/j.tox.2006.10.003. Epub 2006 Oct 13.
Renal cell carcinoma is the most common neoplasm occurring in the kidney and is largely resistant to current chemotherapy. Understanding the mechanisms involved in renal carcinoma cell death may lead to novel and more effective therapies. In Cak(i)-1 renal cancer cells, using phosphatidylserine externalization as a marker of apoptosis, the anti-cancer drugs 5-fluorouracil (5-FU), and its pro-drugs, doxifluridine (Dox) and floxuridine (Flox) proceeds via a caspase-dependent mechanism. In contrast, phosphatidylserine externalization produced by staurosporine in the renal cancer cell lines Cak(i)-1 and A-498 proceeds via a caspase-independent mechanism. That is, the pan caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) did not ameliorate annexin V binding, cell shrinkage or changes in nuclear morphology. Subsequent experiments were conducted to determine mediators of phosphatidylserine externalization, using annexin V binding, when caspases were inhibited. Prior treatment of A-498 cells with cathepsin B (CA74 methyl ester), cathespsin D (pepstatin A) or calpain inhibitors (calpeptin, E64d) in the presence or absence of ZVAD did not ameliorate annexin V binding. The endonuclease inhibitor aurintricarboxylic acid (ATA), phospholipase A(2) inhibitor bromoenol lactone (BEL), protein synthesis inhibitor cycloheximide (CH) and chloride channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) all had no effect on staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. We also modulated sphingomyelin and the de novo pathways of ceramide synthesis and found no amelioration of staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. These results indicate that 5-FU, Dox and Flox induce externalization of phosphatidylserine during apoptosis in Cak(i)-1 renal cancer cells primarily through a caspase-dependent mechanism and that externalization of phosphatidylserine during apoptosis produced by staurosporine in the renal cancer cell line A-498 is independent of many of the common signaling pathways known to be involved in this process.
肾细胞癌是肾脏中最常见的肿瘤，并且对当前的化疗大多具有抗性。了解参与肾癌细胞死亡的机制可能会带来新的、更有效的治疗方法。在Cak(i)-1肾癌细胞中，以磷脂酰丝氨酸外化作为细胞凋亡的标志物，抗癌药物5-氟尿嘧啶（5-FU）及其前体药物多西氟啶（Dox）和氟尿苷（Flox）通过半胱天冬酶依赖性机制发挥作用。相比之下，在肾癌细胞系Cak(i)-1和A-498中，星形孢菌素诱导的磷脂酰丝氨酸外化通过半胱天冬酶非依赖性机制进行。也就是说，泛半胱天冬酶抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（ZVAD）并不能改善膜联蛋白V结合、细胞皱缩或核形态的变化。随后进行了实验，以确定在半胱天冬酶被抑制时，使用膜联蛋白V结合来检测磷脂酰丝氨酸外化的介质。在存在或不存在ZVAD的情况下，用组织蛋白酶B（CA74甲酯）、组织蛋白酶D（胃蛋白酶抑制剂A）或钙蛋白酶抑制剂（钙蛋白酶抑制剂、E64d）预先处理A-498细胞，均不能改善膜联蛋白V结合。核酸内切酶抑制剂金精三羧酸（ATA）、磷脂酶A(2)抑制剂溴烯醇内酯（BEL）、蛋白质合成抑制剂环己酰亚胺（CH）以及氯离子通道阻滞剂氟尼辛（NFA）和5-硝基-2-(3-苯丙基氨基)苯甲酸（NPPB），无论在存在或不存在ZVAD的情况下，对星形孢菌素诱导的A-498细胞膜联蛋白V结合均无影响。我们还调节了鞘磷脂和神经酰胺合成的从头途径，发现在存在或不存在ZVAD的情况下，对星形孢菌素诱导的A-498细胞膜联蛋白V结合均无改善作用。这些结果表明，5-FU、Dox和Flox在Cak(i)-1肾癌细胞凋亡过程中主要通过半胱天冬酶依赖性机制诱导磷脂酰丝氨酸外化，并且在肾癌细胞系A-498中，星形孢菌素诱导的凋亡过程中磷脂酰丝氨酸外化独立于许多已知参与此过程的常见信号通路。
