The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line
The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line
复制标题
DOI：
10.1158/1535-7163.mct-05-0243
复制
发表时间：
2006-02-01
影响因子：
5.7
通讯作者：
Goldman, DI
中科院分区：
文献类型：
--
作者：
Chattopadhyay, S;Zhao, RB;Goldman, DI
培美曲塞是新一代叶酸拮抗剂，是还原型叶酸载体（RFC）的良好底物，最近被批准用于间皮瘤和非小细胞肺癌的治疗。为了探索载体对培美曲塞活性的影响，通过诱变和PT 632选择压力使RFC在HCT-15结肠癌细胞中失活。获得了在氨基酸401处具有甘氨酸至精氨酸取代的克隆（PT 1），导致RFC功能丧失。PT 1细胞对PT 632（178倍）、甲氨蝶呤（4倍）和ZD 1694（Tomudex，雷替曲塞; 20倍）具有耐药性，但在25 nmol/L 5-甲酰四氢叶酸中生长时，对培美曲塞具有3倍的间接敏感性。野生型RFC转染的PT 1细胞具有与野生型HCT-15细胞相当的抗叶酸剂敏感性，表明RFC突变是耐药性的唯一基础。通过放射性标记细胞内叶酸或酶结合试验分别测量，PT 1细胞中的叶酸池收缩了32%或60%。这反映在对曲美蝶呤的显著（6.5倍）附带敏感性中。PT 1细胞中培美曲塞的初始摄取显著降低（接近85%），但2小时和6天后，细胞内培美曲塞水平分别增加至野生型细胞的60%和70%。基于核苷保护分析，在5-甲酰四氢叶酸盐中生长的PT 1细胞中，培奈曲塞对甘氨酰胺核糖核苷酸转化酶的抑制作用增加，对胸苷酸合成酶的抑制程度较小。因此，RFC功能丧失导致HCT-15细胞对培美曲塞产生附带敏感性，这可能是由于细胞叶酸池收缩导致培美曲塞多聚谷氨酰化部分保留和靶酶抑制增加。
Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD 1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced (similar to 85%) but intracellular pemetrexed levels increased to similar to 60% and similar to 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pernetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition.