Genetic and biochemical modulation of 5-fluorouracil through the overexpression of thymidine kinase: an in-vitro study
Genetic and biochemical modulation of 5-fluorouracil through the overexpression of thymidine kinase: an in-vitro study
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DOI：
10.1097/01.cad.0000198914.83195.61
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发表时间：
2006-04-01
影响因子：
2.3
通讯作者：
Ciccolini, Joseph
中科院分区：
文献类型：
--
作者：
Fanciullino, Raphaelle;Evrard, Alexandre;Ciccolini, Joseph
前药5-氟尿嘧啶（5-FU）转化为细胞毒性代谢物后发挥其抗增殖作用。我们之前证明了5-FU的抗癌作用可以通过提高癌细胞中的胸腺嘧啶磷酸化酶（TP）活性来增强，这是DNA途径的第一步，产生关键的抗胸腺嘧啶合成酶（TS）氟脱氧尿苷单磷酸（FdUMP）代谢物。在本研究中，我们进一步研究了过表达癌细胞胸苷激酶（TK）在多大程度上可以优化5-FU活性，这是DNA途径的第二步，迄今为止有争议的数据已发表。此外，筛选可能有助于5-FU激活的生化调节剂也进行了。设计携带病毒和人cDNA的载体，稳定转染人HT29细胞系，获得tk过表达的结直肠细胞。随后进行抗增殖试验，以评估细胞对5-FU的敏感性变化，并进行代谢监测，以跟踪药物激活和细胞摄取后的湿度形成。最后，将TS抑制作为药理学终点进行评估。结果表明，TK的过表达导致我们的模型显着脱敏。TK活性水平与5-FU抗增殖作用呈负相关（r(2)=0.87），活性越高，敏感性越低。在各种被筛选为可能的调节剂的药物中，只有那些参与TP活性的药物被证明通过优化FdUMP的形成来增强5-FU的功效。相反，在我们的模型中，遗传上增加TK活性并没有改变5-FU激活途径和随后的TS抑制。因此，我们的研究结果表明TK不是产生抗ts驼峰的限制步骤，过表达TK的肿瘤细胞可能会抵抗基于5- fu的化疗。
The pro-drug 5-fluorouracil (5-FU) exerts its anti-proliferative action after conversion into cytotoxic metabolites. We previously demonstrated that the anti-cancer action of 5-FU could be enhanced by boosting thymidine phosphorylase (TP) activity in cancer cells, the first step of the DNA pathway, that yields the critical anti-thymidylate synthase (TS) fluorodeoxyuridine monophosphate (FdUMP) metabolite. In the present study, we further studied to what extent 5-FU activity could be optimized by overexpressing cancer cell thymidine kinase (TK), the second step of the DNA pathway, for which controversial data have been published so far. Additionally, screening of biochemical modulators likely to contribute to 5-FU activation was also carried out. TK-overexpressing colorectal cells were obtained after designing vectors harboring viral and human cDNA, and performing stable transfection in the human HT29 cell line. Anti-proliferative assays were subsequently performed so as to evaluate change in cell sensitivity to 5-FU, and metabolism monitoring was carried out to follow drug activation and HUMID formation after cellular uptake. Finally, TS inhibition was assessed as a pharmacological endpoint. Results showed that overexpression of TK led to a marked desensitization of our model. A negative correlation (r(2)=0.87) was found between the level of TK activity and 5-FU anti-proliferative action - the higher the activity, the lower the sensitivity. Of the various drugs screened as putative modulators, only those involved in TP activity proved to enhance 5-FU efficacy via optimized FdUMP formation. Conversely, genetically increasing TK activity did not modify 5-FU activation pathway nor subsequent TS inhibition in our model. Therefore, our results indicate that TK is not a limiting step in the production of anti-TS HUMP and that tumor cells overexpressing TK are likely to resist 5-FU-based chemotherapies.