Concentrative Transport of Antifolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer

Abstract

The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO-buffered medium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na/H exchanger(s); it was eliminated by inhibitors of Na/H exchanger (s) or Na/K ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds

“…PCFT has recently emerged as a predictive parameter in MPM patients treated with pemetrexed-based chemotherapeutic regimens, since low PCFT expression was associated with shorter PFS and OS. 13 PCFT is the main transporter mediating pemetrexed influx, with remarkable transport Km values of 0.2–0.8 μM at acidic pH, 42 and its expression has been linked to growth-inhibitory activity of pemetrexed after transfection into a folate transporter-null cell variant. 43 We consistently observed that PCFT silencing increased the IC 50 values of pemetrexed in our MPM cell lines.…”

A guide to plasma membrane solute carrier proteins

“…Polymorphisms of this transporter have been associated with an increase in adverse drug responses [228,229]. SLC46A1 provides an intriguing opportunity to harness its concentrative transport activity in acidic environments, which are often found in the tumor microenvironment [230]. SLC46A1, which is ubiquitously expressed in solid tumors, could in principle afford a more targeted delivery of antifolate drugs to tumor cells [230].…”

“…SLC46A1 provides an intriguing opportunity to harness its concentrative transport activity in acidic environments, which are often found in the tumor microenvironment [230]. SLC46A1, which is ubiquitously expressed in solid tumors, could in principle afford a more targeted delivery of antifolate drugs to tumor cells [230]. Expression of SLC19A3 in cancerous cells has been shown to be both increased and decreased and further studies are needed to elucidate the role SLC19A3 plays in cancer cells [224,231].…”

Transcriptomic profiling of hepatic tissues for drug metabolism genes in nonalcoholic fatty liver disease: A study of human and animals

“…Losing the function of the proton-coupled folate transporter protein caused hereditary folate malabsorption ( 49 ). In addition, SLC46A1 transports folic acid, methotrexate, and pemetrexed ( 34 – 36 ). The lysosomal copper transporter SLC46A3 was localized to the lysosome, modulating intracellular copper levels ( 26 ).…”