Lam Wing, Leung Chung-Hang, Bussom Scott, Cheng Yung-Chi
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
Mol Pharmacol. 2007 Sep;72(3):536-44. doi: 10.1124/mol.106.033472. Epub 2007 Jun 12.
beta-L-Dioxolane-cytidine (L-OddC, Troxacitabine, BCH-4556), a novel L-configuration deoxycytidine analog, is under clinical trials for treating cancer. The cytotoxicity of L-OddC is dependent on the amount of the triphosphate form (L-OddCTP) in nuclear DNA. Phosphoglycerate kinase (PGK), a downstream protein of hypoxia-inducible-factor-1alpha (HIF-1alpha), is responsible for the phosphorylation of the diphosphate to the triphosphate of L-OddC. In this study, we studied the impact of hypoxia on the metabolism and the cytotoxicity of L-OddC and beta-d-2',2'-difluorodeoxycytidine (dFdC) in several human tumor cell lines including HepG2, Hep3B, A673, Panc-1, and RKO. Hypoxic treatment induced the protein expression of PGK 3-fold but had no effect on the protein expression of APE-1, dCK, CMPK, and nM23 H1. Hypoxic treatment increased L-OddCTP formation and incorporation of L-OddC into DNA, but it decreased the uptake and incorporation of dFdC, which correlated with the reduction of hENT1, hENT2, and hCNT2 expression. Using a clonogenic assay, hypoxic treatment of cells made them 2- to 3-fold more susceptible to L-OddC but not to dFdC after exposure to drugs for one generation. Dimethyloxallyl glycine enhanced the cytotoxicity of L-OddC but not dFdC in Panc-1 cells under normoxic conditions. Overexpression or down-regulation of PGK using transient transfection of pcDNA5-PGK or inducible shRNA in RKO cells affected the cytotoxicity of L-OddC but not that of dFdC. The knockdown of HIF-1alpha in inducible shRNA in RKO cells reduced the cytotoxicity of L-OddC but not dFdC under hypoxic conditions. In conclusion, hypoxia is an important factor that may potentiate the activity of L-OddC.
β-L-二氧戊环胞苷（L-OddC，曲沙他滨，BCH-4556）是一种新型的L构型脱氧胞苷类似物，正在进行治疗癌症的临床试验。L-OddC的细胞毒性取决于核DNA中三磷酸形式（L-OddCTP）的含量。磷酸甘油酸激酶（PGK）是缺氧诱导因子-1α（HIF-1α）的下游蛋白，负责将L-OddC的二磷酸磷酸化为三磷酸。在本研究中，我们研究了缺氧对几种人类肿瘤细胞系（包括HepG2、Hep3B、A673、Panc-1和RKO）中L-OddC和β-d-2'，2'-二氟脱氧胞苷（dFdC）代谢及细胞毒性的影响。缺氧处理使PGK蛋白表达增加3倍，但对APE-1、dCK、CMPK和nM23 H1的蛋白表达没有影响。缺氧处理增加了L-OddCTP的形成以及L-OddC掺入DNA，但降低了dFdC的摄取和掺入，这与hENT1、hENT2和hCNT2表达的降低相关。使用克隆形成试验，在细胞暴露于药物一代后，缺氧处理使它们对L-OddC的敏感性提高2至3倍，但对dFdC不敏感。在常氧条件下，二甲基草酰甘氨酸增强了Panc-1细胞中L-OddC的细胞毒性，但对dFdC没有影响。在RKO细胞中使用pcDNA5-PGK瞬时转染或诱导性shRNA过表达或下调PGK影响了L-OddC的细胞毒性，但对dFdC没有影响。在RKO细胞中使用诱导性shRNA敲低HIF-1α降低了缺氧条件下L-OddC的细胞毒性，但对dFdC没有影响。总之，缺氧是可能增强L-OddC活性的一个重要因素。
