Functional characterization of RFC and PCFT for antifolates accumulation in non-small cell lung cancer cells .

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 K m value of PCFT for pemetrexed at pH 7.4 was more than 10 times higher than that at pH 6.5. Interestingly, we found 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 IC 50 value of pemetrexed at pH 7.4 being one-third of that at pH 6.5. In addition, the in vivo study indicated 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 RFC and PCFT on antifolates accumulation in NSCLC is necessary for new drug designs. By using RFC- or PCFT-expressing NSCLC cell models, we found that both RFC and PCFT were important for antifolates accumulation in NSCLC, rather than only PCFT playing a dominant role. 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.