Michele Visentin

Mechanisms of Membrane Transport of Folates into Cells and Across Epithelia

Until recently, the transport of folates into cells and across epithelia has been interpreted primarily within the context of two transporters with high affinity and specificity for folates, the reduced folate carrier and the folate receptors. However, there were discrepancies between the properties of these transporters and characteristics of folate transport in many tissues, most notably the intestinal absorption of folates, in terms of pH dependency and substrate specificity. With the recent cloning of the proton-coupled folate transporter (PCFT) and the demonstration that this transporter is mutated in hereditary folate malabsorption, an autosomal recessive disorder, the molecular basis for this low-pH transport activity is now understood. This review focuses on the properties of PCFT and briefly addresses the two other folate-specific transporters along with other facilitative and ATP-binding cassette (ABC) transporters with folate transport activities. The role of these transporters in the vectorial transport of folates across epithelia is considered.

The Intestinal Absorption of Folates

The properties of intestinal folate absorption were documented decades ago. However, it was only recently that the proton-coupled folate transporter (PCFT) was identified and its critical role in folate transport across the apical brush-border membrane of the proximal small intestine established by the loss-of-function mutations identified in the PCFT gene in subjects with hereditary folate malabsorption and, more recently, by the Pcft-null mouse. This article reviews the current understanding of the properties of PCFT-mediated transport and how they differ from those of the reduced folate carrier. Other processes that contribute to the transport of folates across the enterocyte, along with the contribution of the enterohepatic circulation, are considered. Important unresolved issues are addressed, including the mechanism of intestinal folate absorption in the absence of PCFT and regulation of PCFT gene expression. The impact of a variety of ions, organic molecules, and drugs on PCFT-mediated folate transport is described.

Substrate- and pH-specific antifolate transport mediated by organic anion-transporting polypeptide 2B1 (OATP2B1-SLCO2B1).

Human organic anion-transporting polypeptide (OATP) 2B1 (OATP-B; SLCO2B1) is expressed in the apical membrane of the small intestine and the hepatocyte basolateral membrane and transports structurally diverse organic anions with a wide spectrum of pH sensitivities. This article describes highly pH-dependent OATP2B1-mediated antifolate transport and compares this property with that of sulfobromophthalein (BSP), a preferred OATP2B1 substrate. At pH 5.5 and low substrate concentrations (∼2.5 μM), only [3H]pemetrexed influx [in contrast to methotrexate (MTX), folic acid, and reduced folates] could be detected in OATP2B1-transfected HeLa R1-11 cells that lack endogenous folate-specific transporters. Influx was optimal at pH 4.5 to 5.5, falling precipitously with an increase in pH >6.0; BSP influx was independent of pH. Influx of both substrates at low pH was markedly inhibited by the proton ionophore 4-(trifluoromethoxy)phenylhydrazone; BSP influx was also suppressed at pH 7.4. At 300 μM MTX, influx was one-third that of pemetrexed; influx of folic acid, (6S)5-methyltetrahydrofolate, or (6S)5-formyltetrahydrofolate was not detected. There were similar findings in OATP2B1-expressing Xenopus laevis oocytes. The pemetrexed influx Km was ∼300 μM; the raltitrexed influx Ki was ∼70 μM at pH 5.5. Stable expression of OAPT2B1 in HeLa R1-11 cells resulted in substantial raltitrexed, but modest pemetrexed, growth inhibition consistent with their affinities for this carrier. Hence, OATP2B1 represents a low-affinity transport route for antifolates (relative affinities: raltitrexed > pemetrexed > MTX) at low pH. In contrast, the high affinity of this transporter for BSP relative to antifolates seems to be intrinsic to its binding site and independent of the proton concentration.

Augmentation of reduced folate carrier-mediated folate/antifolate transport through an antiport mechanism with 5-aminoimidazole-4-carboxamide riboside monophosphate

5-Aminoimidazole-4-carboxamide riboside (AICAR), an agent with diverse pharmacological properties, augments transport of folates and antifolates. This report further characterizes this phenomenon and defines the mechanism by which it occurs. Exposure of HeLa cells to AICAR resulted in augmentation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate initial rates and net uptake in cells that express the reduced folate carrier (RFC). This did not occur in cells that express only the proton-coupled folate transporter and accumulated folates by this mechanism. Transport stimulation correlated with the accumulation of 5-aminoimidazole-4-carboxamide ribotide monophosphate (ZMP), the monophosphate derivative of AICAR, within cells as established by liquid chromatography. When ZMP formation was blocked with 5-iodotubercidin, an inhibitor of adenosine kinase, folate transport stimulation by AICAR was absent. When cells first accumulated ZMP and were then exposed to 5-iodotubercidin or AICAR-free buffer, the ZMP level markedly decreased and folate transport stimulation was abolished. Extracellular ZMP inhibited RFC-mediated folate influx, and the presence of intracellular ZMP correlated with inhibition of folate efflux. The data indicate that intracellular ZMP trans-stimulates folate influx and inhibits folate efflux, which, together, produce a marked augmentation in the net cellular folate level. This interaction among ZMP, folates, and RFC, a folate/organic phosphate antiporter, is consistent with a classic exchange reaction. The transmembrane gradient for one transport substrate (ZMP) drives the uphill transport of another (folate) via a carrier used by both substrates, a phenomenon intrinsic to the energetics of RFC-mediated folate transport.

Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1)

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [(3)H]methotrexate and [(3)H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [(3)H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carriers mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.

Inhibition of the Proton-Coupled Folate Transporter (PCFT-SLC46A1) by Bicarbonate and Other Anions

The proton-coupled folate transporter (PCFT) plays a key role in intestinal folate absorption, and loss-of-function mutations in the gene encoding this transporter are the molecular basis for hereditary folate malabsorption. Using a stable transfectant with high expression of PCFT, physiologic levels of bicarbonate produced potent and rapidly reversible inhibition of PCFT-mediated transport at neutral pH. Bisulfite and nitrite also inhibited PCFT function at neutral pH, whereas sulfate, nitrate, and phosphate had no impact at all. At weakly acidic pH (6.5), bisulfite and nitrite exhibited much stronger inhibition of PCFT-mediated transport, whereas sulfate and nitrate remained noninhibitory. Inhibition by bisulfite and nitrite at pH 6.5 was associated with a marked decrease in the influx Vmax and collapse of the transmembrane proton gradient attributed to the diffusion of the protonated forms into these cells. Monocarboxylates such as pyruvate and acetate also collapsed the pH gradient and were also inhibitory, whereas citrate and glycine neither altered the proton gradient nor inhibited PCFT-mediated transport. These observations add another dimension to the unfavorable pH environment for PCFT function in systemic tissues: the presence of high concentrations of bicarbonate.

Drug interactions among the epidermal growth factor receptor inhibitors, other biologics and cytotoxic agents

The epidermal growth factor receptor (EGFR) signalling pathway is a key element in the growth of several epithelial malignancies. Small molecules tyrosine kinase inhibitors (TKIs) and anti-EGFR monoclonal antibodies (mAbs) prevent the phosphorylation of the receptor, leading to cell cycle arrest at G(1) phase, apoptosis, inhibition of angiogenesis and metastasis. To increase the antitumoral effects of EGFR inhibitors (EGFRIs), a number of combinatory regimens have been evaluated and planned with standard cytotoxic drugs and/or inhibitors of EGFR complementary pathways such as mTOR, VEGF and Ras/Raf/ERK. Compared to EGFRI monotherapy, the combination approach is a promising strategy to improve tumor response and survival. However, pharmacokinetic (absorption, distribution, metabolism and excretion) and pharmacodynamic drug interactions can occur, affecting the outcome. Pharmacokinetics of TKIs can be affected by drugs used in combination: conversely, pharmacokinetic interactions have not been reported for EGFR mAbs. Potential pharmacokinetic interactions occur between EGFRIs and other factors such as food and hydrocarbons in tobacco smoke were also considered. EGFRIs are characterized by a number of pharmacodynamic interactions that must be taken into consideration to avoid adverse events, to increase antitumoral activity, and define potential new strategies for developing efficient combination regimens. In this context, treatment schedule and drug sequence appear to be particularly relevant for combination regimens with EGFRIs. Improved molecular characterisation of the EGFR pathway and its complementary pathways in tumor cells is required to better define predictive pharmacokinetic and pharmacodynamic biomarkers for optimum treatment outcome.

Polymorphic thymidylate synthase gene impacts on overall survival of patients with epithelial ovarian cancer after platinum-based chemotherapy

AIM High levels of TS have been associated with a worse clinical outcome in several cancers including epithelial ovarian cancer (EOC). The TS gene (TYMS) is highly polymorphic and has an effect on mRNA/protein expression. MATERIALS & METHODS Six TYMS polymorphisms were investigated for overall survival (OS) in 216 EOC patients: TYMS 1494ins/del, TSER (variable number of tandem repeats of 28 bp), TSER G>C, TYMS 1053C>T, TYMS IVS6-68C>T and TYMS 1122A>G. RESULTS In a multivariate analysis, TYMS 1494 del/del genotype was associated with a significant increased OS compared with the ins/ins genotype (hazard ratio: 0.36; 95% CI: 0.16-0.82, p = 0.01). Similar results were obtained for the mutant genotypes TYMS 1053TT and TYMS IVS6-68TT. The event-free survival was significantly higher in TYMS 1053TT patients compared with wild-type patients (p = 0.05). CONCLUSION TYMS 1494ins/del, 1053C>T and IVS6-68C>T polymorphisms can be prognostic markers for OS in patients with EOC, independently from stage at diagnosis, median age and tumor histotype.

Effects of Farnesoid X Receptor Activation on Arachidonic Acid Metabolism, NF-kB Signaling, and Hepatic Inflammation

Inflammation has a recognized role in nonalcoholic fatty liver disease (NAFLD) progression. In the present work, we studied the effect of high-fat diet (HFD) on arachidonic acid metabolism in the liver and investigated the role of the farnesoid X receptor (FXR, NR1H4) in eicosanoid biosynthetic pathways and nuclear factor κ light-chain enhancer of activated B cells (NF-kB) signaling, major modulators of the inflammatory cascade. Mice were fed an HFD to induce NAFLD and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analyses were performed on liver tissue. Eicosanoid levels were measured from serum and urine samples. The molecular mechanism underlying the effect of FXR activation on arachidonic acid metabolism and NF-kB signaling was studied in human liver Huh7 cells and primary cultured hepatocytes. NAFLD was characterized by higher (∼25%) proinflammatory [leukotrienes (LTB4)] and lower (∼3-fold) anti-inflammatory [epoxyeicosatrienoic acids (EETs)] eicosanoid levels than in chow mice. OCA induced the expression of several hepatic cytochrome P450 (P450) epoxygenases, the enzymes responsible for EET synthesis, and mitigated HFD-induced hepatic injury. In vitro, induction of CYP450 epoxygenases was sufficient to inhibit NF-kB signaling and cell migration. The CYP450 epoxygenase pan-inhibitor gemfibrozil fully abolished the protective effect of OCA, indicating that OCA-mediated inhibition of NF-kB signaling was EET-dependent. In summary, NAFLD was characterized by an imbalance in arachidonate metabolism. FXR activation reprogramed arachidonate metabolism by inducing P450 epoxygenase expression and EET production. In vitro, FXR-mediated NF-kB inhibition required active P450 epoxygenases.

Abstract 1784: The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Pralatrexate, the 10-propargyl-10-deaza analog of aminopterin, is a much superior substrate for the reduced folate carrier and folylpolyglutamate synthetase than methotrexate (MTX) resulting in the rapid build-up and retention of high levels of the polyglutamate derivatives of this agent within cells. In HeLa cells in vitro, the growth inhibitory effect of a 6h exposure to pralatrexate is two orders of magnitude greater than that of MTX. Yet the drug can be administered weekly at similar doses than MTX, consistent with a high level of selectivity. Nonetheless, mucosites can result in dosage reductions. This has led to the application of “rescue” regimens with the natural isomer of 5-formyltetrahydrofolate, (6S)5-formylTHF. Purpose: To investigate the impact of (6S)5-formylTHF on the antitumor activity of pralatrexate in HeLa cells in vitro, simulating the exposures to this folate as occurs in the clinical setting. These properties were compared to those of MTX. Experimental design: HeLa cells were grown with 25 nM (6S)5-formylTHF as the folate growth source. Experiments evaluated the impact of a 24h exposure to different concentration of (6S)5-formylTHF varying intervals prior to, or after, a 6h exposure to antifolate. The impact on the accumulation of [3H](6S)5-formylTHF and [3H]antifolate was also assessed. Results: A 24h interval between antifolate, followed by (6S)5-formylTHF, was sufficient to sustain the full activities of the antifolates up to a (6S)5-formylTHF level of 4 µM. A 72h interval was required between (6S)5-formylTHF, followed by the antifolate, to sustain full antifolate activities. HeLa cells were then exposed to 4 µM (6S)5-formylTHF for 24h weekly X 4; a concentration of the folate in excess of what is achieved in “rescue” regimens. This had no significant effect on the pralatrexate IC50; however, the MTX IC50 increased 2.5-fold. The four cycles of exposure to 4 µM [3H](6S)5-formylTHF resulted in a 16% increase in the tritiated intracellular folate level (P=0.02) as compared to cells growing continuously in 25 nM [3H](6S)5-formylTHF.There was no significant decrease in total intracellular pralatrexate; there was a 15% decrease in total intracellular MTX (P=0.004). Conclusions: Exposure of HeLa cells to (6S)5-formylTHF either before or after pralatrexate or MTX, simulating the sequence of administration in “rescue” regimens, and at concentrations in excess of what is achieved clinically, had no effect on pralatrexate activity but decreased the activity of MTX. This cumulative effect was associated with a small increase in the intracellular (6S)5-formylTHF level and a small decrease in total intracellular MTX. The data suggests that standard “rescue” regimens should have little or no impact on the antitumor activity of pralatrexate. Citation Format: Michele Visentin, Ersin Selcuk Unal, I David Goldman. The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2014-1784