Zhao Rongbao, Gao Feng, Hanscom Marie, Goldman I David
Department of Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Clin Cancer Res. 2004 Jan 15;10(2):718-27. doi: 10.1158/1078-0432.ccr-1066-03.
Whereas the major folate transporter, the reduced folate carrier (RFC), has a physiological pH optimum, transport activities for folates and antifolates have been detected with low pH optima. Because the interstitial pH in solid tumors is generally acidic, the mechanisms by which antifolates are transported at low pH could be an important determinant of drug activity under these conditions. The current study quantitated the low pH methotrexate (MTX) transport activity in human solid tumor cell lines from the National Cancer Institute tumor panel and other sources. MTX influx at pH 5.5 was equal to, or greater than, influx at pH 7.4 in 29 of 32 cell lines. To assess the role of RFC in transport at low pH in one of these cell lines, a HeLa clonal line (R5) was selected for MTX resistance due to a genomic deletion of the carrier gene. MTX influx was depressed by 70% in R5 versus wild-type HeLa cells at pH 7.4. At pH 6.5, influx in these two lines was similar; as the pH was decreased to 5.5 influx increased in both cell lines. Similarly, whereas net MTX uptake over 1 h was markedly decreased in R5 cells at pH 7.4, net uptake in HeLa and R5 cells was comparable at pH 6.5. Also, as compared with MCF7 breast cancer cells, MTX uptake was markedly decreased at pH 7.4, but only minimally at pH 6.5, in the MDA-MB-231 human breast cancer cell line that lacks RFC expression. When grown with folic acid (2 micro M) at pH 7.4, the IC(50) for MTX was 14-fold higher in R5 as compared with wild-type HeLa cells; the difference was only 4-fold when cells when grown at pH 6.9; the IC(50)s were identical at this pH when the medium folate was 25 nM 5-formyltetrahydrofolate. These data demonstrate that transport activity at low pH is prevalent in human solid tumors, is RFC-independent in R5 cells and MDA-MB-231 breast cancer cells, and can preserve MTX activity in the absence of RFC at an acidic pH relevant to solid tumors in vivo.
虽然主要的叶酸转运体——还原型叶酸载体（RFC）具有生理最适pH值，但已检测到叶酸和抗叶酸药物在低pH最适值时的转运活性。由于实体瘤中的间质pH通常呈酸性，抗叶酸药物在低pH下的转运机制可能是这些条件下药物活性的重要决定因素。本研究对来自美国国立癌症研究所肿瘤库及其他来源的人类实体瘤细胞系中的低pH甲氨蝶呤（MTX）转运活性进行了定量分析。在32个细胞系中的29个中，pH 5.5时的MTX内流等于或大于pH 7.4时的内流。为了评估RFC在其中一个细胞系低pH转运中的作用，选择了一个因载体基因基因组缺失而对MTX耐药的HeLa克隆系（R5）。在pH 7.4时，R5细胞中的MTX内流比野生型HeLa细胞降低了70%。在pH 6.5时，这两个细胞系中的内流相似；随着pH降至5.5，两个细胞系中的内流均增加。同样，虽然在pH 7.4时R5细胞中1小时内MTX的净摄取量显著降低，但在pH 6.5时HeLa和R5细胞中的净摄取量相当。此外，与MCF7乳腺癌细胞相比，缺乏RFC表达的MDA-MB-231人乳腺癌细胞系在pH 7.4时MTX摄取显著降低，但在pH 6.5时仅略有降低。当在pH 7.4下用叶酸（2 μM）培养时，R5细胞中MTX的IC50比野生型HeLa细胞高14倍；当细胞在pH 6.9下培养时，差异仅为4倍；当培养基叶酸为25 nM 5-甲酰四氢叶酸时，在该pH下IC50相同。这些数据表明，低pH转运活性在人类实体瘤中普遍存在，在R5细胞和MDA-MB-231乳腺癌细胞中与RFC无关，并且在体内与实体瘤相关的酸性pH条件下，在没有RFC的情况下可以保持MTX活性。
