Translational Medicine Research Center, Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, Hangzhou, China (Y.W., J.L., M.B., Z.M., S.M., S.Z.); Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China (J.L., H.J.); The Fourth College of Clinical Medicine, Zhejiang Chinese Medical University, Hangzhou, China (Y.X.); and Shanghai AB SCIEX Analytical Instrument Trading Co., Ltd., Shanghai, China (D.L.).
Translational Medicine Research Center, Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, Hangzhou, China (Y.W., J.L., M.B., Z.M., S.M., S.Z.); Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China (J.L., H.J.); The Fourth College of Clinical Medicine, Zhejiang Chinese Medical University, Hangzhou, China (Y.X.); and Shanghai AB SCIEX Analytical Instrument Trading Co., Ltd., Shanghai, China (D.L.)
Drug Metab Dispos. 2024 Oct 16;52(11):1332-1344. doi: 10.1124/dmd.124.001872.
Antifolates are important for chemotherapy in non-small cell lung cancer (NSCLC). They mainly rely on reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) to enter cells. PCFT is supposed to be the dominant transporter of the two in tumors, as it operates optimally at acidic pH and has limited transport activity at physiological pH, whereas RFC operates optimally at neutral pH. In this study, we found RFC showed a slightly pH-dependent uptake of antifolates, with similar affinity values at pH 7.4 and 6.5. PCFT showed a highly pH-dependent uptake of antifolates, with an optimum pH of 6.0 for pemetrexed and 5.5 for methotrexate. The Michaelis-Menten constant ( ) value of PCFT for pemetrexed at pH 7.4 was more than 10 times higher than that at pH 6.5. Interestingly, we found that antifolate accumulations mediated by PCFT at acidic pH were significantly affected by the efflux transporter, breast cancer resistance protein (BCRP). The highest pemetrexed concentration was observed at pH 7.0-7.4 after a 60-minute accumulation in PCFT-expressing cells, which was further evidenced by the cytotoxicity of pemetrexed, with the value of pemetrexed at pH 7.4 being one-third of that at pH 6.5. In addition, the in vivo study indicated that increasing PCFT and RFC expression significantly enhanced the antitumor efficacy of pemetrexed despite the high expression of BCRP. These results suggest that both RFC and PCFT are important for antifolates accumulation in NSCLC, although there is an acidic microenvironment and high BCRP expression in tumors. SIGNIFICANCE STATEMENT: Evaluating the role of reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) on antifolates accumulation in non-small cell lung cancer (NSCLC) is necessary for new drug designs. By using cell models, we found both RFC and PCFT were important for antifolates accumulation in NSCLC. Breast cancer resistance protein (BCRP) significantly affected PCFT-mediated antifolates accumulation at acidic pH but not RFC-mediated pemetrexed accumulation at physiological pH. High expression of PCFT or RFC enhanced the cytotoxicity and antitumor effect of pemetrexed.
叶酸拮抗剂在非小细胞肺癌（NSCLC）的化疗中非常重要。它们主要依靠还原叶酸载体（RFC）和质子偶联叶酸转运体（PCFT）进入细胞。PCFT 被认为是两种转运体在肿瘤中的主要转运体，因为它在酸性 pH 下最佳运作，在生理 pH 下转运活性有限，而 RFC 在中性 pH 下最佳运作。在这项研究中，我们发现 RFC 对叶酸拮抗剂的摄取表现出轻微的 pH 依赖性，在 pH 7.4 和 6.5 时具有相似的亲和力值。PCFT 对叶酸拮抗剂的摄取表现出高度的 pH 依赖性，培美曲塞的最佳 pH 为 6.0，甲氨蝶呤的最佳 pH 为 5.5。PCFT 在 pH 7.4 时对培美曲塞的米氏常数（ ）值比 pH 6.5 时高出 10 多倍。有趣的是，我们发现酸性 pH 下由 PCFT 介导的叶酸拮抗剂积累会受到外排转运蛋白乳腺癌耐药蛋白（BCRP）的显著影响。在 PCFT 表达细胞中孵育 60 分钟后，在酸性 pH 值下观察到最高的培美曲塞浓度，这进一步证明了培美曲塞的细胞毒性，pH 值为 7.4 时的 值是 pH 值为 6.5 时的三分之一。此外，体内研究表明，尽管 BCRP 高表达，增加 PCFT 和 RFC 的表达可显著增强培美曲塞的抗肿瘤疗效。这些结果表明，尽管肿瘤中存在酸性微环境和高 BCRP 表达，但 RFC 和 PCFT 对 NSCLC 中叶酸拮抗剂的积累都很重要。
