Reduced Folate Carrier Levels Research Articles

Significance of thymidylate synthase expression for resistance to pemetrexed in pulmonary adenocarcinoma

Pemetrexed (PEM), a multi-targeted antifolate, has promising clinical activity in non-squamous non-small cell lung cancer. However, the majority of patients eventually acquire resistance to PEM. To evaluate the resistant mechanisms, the A549 lung adenocarcinoma cell line was exposed to stepwise increasing PEM concentrations of 1.6, 6.4 and 16 μM to establish three PEM-resistant lung cancer cell lines, A549/PEM-1.6, -6.4 and -16. Growth inhibition was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), reduced folate carrier (RFC) and folypoly-γ-glutamate synthetase (FPGS) were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The three A549 cell lines showed more resistance to PEM (3.7-, 17.3− and 38.0-fold, respectively) compared with that of the parental cell line, which also showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine and 5-Fluorouracil (5-FU). TS gene expression was significantly increased in three PEM-resistant cells, relative to that of the parental cells, in a PEM dose-dependent manner. Knockdown of TS expression with siRNA enhanced the cytotoxicity of PEM in A549/PEM-16 cells. By contrast, the levels of RFC and FPGS gene expression in A549/PEM-1.6 and -6.4 cells were significantly decreased, whereas the levels of the two genes were restored in A549/PEM-16 cells. In summary, PEM-resistant A549 cells remained sensitive to docetaxel, vinorelbine and 5-FU. TS expression appeared to be associated with resistance to PEM, which may be a predictive marker for PEM sensitivity in lung adenocarcinoma.

Abstract 873: Acute regulation of human reduced folate carrier by folates as a novel adaptive mechanism to folate deprivation.

Abstract The reduced folate carrier (RFC) is the major tissue folate transporter in mammalian cells. RFC is essential for providing folates for biosynthetic pathways leading to nucleotides and amino acids. Clinically relevant antifolates including methotrexate (Mtx), pemetrexed, and pralatrexate are all substrates for RFC and RFC levels and function are key determinants of antitumor activities. While recent reports have described the development of novel 6-pyrrolo[2,3-d]pyrimidine antifolates with selectivity for cellular uptake and tumor targeting by the proton-coupled folate transporter, RFC levels are nonetheless important determinants of antitumor activities for these agents, by impacting cellular folate pools which compete for polyglutamylation and binding to intracellular enzyme targets. Human RFC is subject to complex transcriptional and posttranscriptional controls. RFC forms homo-oligomers, providing another potential level of regulation, as a dominant-phenotype results with mutant-wild type RFC mixtures. This reflects impaired intracellular trafficking of oligomeric RFC from the endoplasmic reticulum to the plasma membrane surface. To explore the question of whether folates can acutely regulate RFC levels or activity, RFC-null HeLa cells were stably transfected with RFCMyc-his10 in pCDNA3 plasmid. Transfectants were cultured without folate (10 d), then with (6R,S)5-formyltetrahydrofolate (leucovorin (LCV)) from 0.5 to 200 nM (5 d). Cells were assayed for RFC transcripts and total cellular RFC proteins. Membrane surface RFC was measured by sulfo-N-hydroxysuccinimide-SS-biotin labeling and the formation of homo-oligomers was assessed by cross-linking with 1,1-methanediyl bismethanethiosulfonate. Finally, RFC transport was assayed with [3H]Mtx at neutral pH. Levels of RFC transcripts and total cell RFC protein progressively increased with increasing LCV up to 200 nM. Conversely, surface RFC and [3H]Mtx transport were maximal at ∼ 2 nM LCV, then deceased (∼2-3-fold) with increasing LCV. These effects were maximal at 20 nM LCV. RFC plasma membrane targeting was confirmed by immunofluorescence staining and confocal microscopy. RFC protein was completely localized to the plasma membrane at low (≤ 2 nM) LCV concentrations, however, at higher concentrations, significant intracellular RFC was detected. There were no changes in ratios of RFC homo-oligomer over monomer in response to changes in extracellular LCV. Our results suggest a novel acute regulation of RFC in response to changes in extracellular folate concentrations involving (i) increased RFC transcripts and proteins, possibly due to differences in transcript stability, and (ii) increased cytosolic RFC due to impaired intracellular trafficking and membrane targeting. The net effect would be enhanced RFC-mediated folate and antifolate accumulations under conditions of folate deprivation. Citation Format: Zhanjun Hou, Steve Orr, Erika Etnyre, Christina Cherian, Larry Matherly. Acute regulation of human reduced folate carrier by folates as a novel adaptive mechanism to folate deprivation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 873. doi:10.1158/1538-7445.AM2013-873

Abstract 3825: The impact of the reduced folate carrier on the collateral sensitivity of novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective uptake by the proton-coupled folate transporter

Abstract The 6-substituted pyrrolo[2,3-d]pyrimidine thienonyl antifolates with 3 (AG94) or 4 (AG71) carbon bridge lengths represent a new class of agents that inhibit α-glycinamide ribonucleotide (GAR) formyltransferase (GARFTase). AG71 and AG94 are selectively transported by the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), the major facilitative folate transporter in tissues and tumors. PCFT functions optimally at acidic pHs. Transport of AG71 and AG94 by PCFT and the acidic tumor microenvironment may further increase tumor selectivity. Although AG71 and AG94 are not RFC substrates, levels of RFC could nonetheless impact their anti-proliferative effects, via expansion or contraction of intracellular tetrahydrofolate (THF) pools. Studies were performed with wild-type (WT) and methotrexate resistant R5 HeLa cells in which RFC is deleted. In proliferation assays with AG71 and AG94, R5 cells were more sensitive than were WT cells (3.9- and 2.4-fold, respectively). RFC-transfected R5 cells had IC50s for growth inhibition similar to WT cells. To determine the mechanisms of the collateral sensitivities to AG71 and AG94, we measured steady-state intracellular [3H]THF accumulations at a physiologic concentration of [3H]5-formyl THF in WT and R5 cells. R5 cells had contracted intracellular [3H]THF pools compared to WT cells. The collateral antifolate sensitivities in R5 cells were independent of PCFT transport, since there were no differences in transport for AG71 and AG94 at any pH between the sublines. Intracellular THF cofactors may compete at the level of polyglutamylation by folylpolyglutamate synthetase and/or GARFTase binding. When incubated with radiolabeled AG71 and AG94, WT cells accumulated slightly lesser amounts of total drug forms (84 and 76% for AG71 and AG94, respectively) than did R5 cells, most of these as polyglutamates. This difference was greatest for the longest chain-length (Glu4-Glu6) polyglutamates (increased ∼2-fold for both AG71 and AG94). GARFTase activity was measured in WT and R5 cells by an in situ metabolic assay which measures incorporation of [14C]glycine into [14C]formyl GAR. R5 and WT cells were differentially sensitive to GARFTase inhibition by AG71 and AG94 and these differences were lost when RFC expression was restored to R5 cells. In an in vivo efficacy trial with subcutaneous WT and R5 cells in SCID mice, antitumor efficacies of both AG71 and AG94 were increased in R5 cells relative to WT cells and this effect was greater for AG71. These findings suggest that novel antifolates with selective membrane transport by PCFT over RFC would have enhanced activities toward tumors that have lost RFC function, and that levels of RFC may be an important biomarker of chemotherapy efficacy with this new class of solid tumor-selective drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3825. doi:1538-7445.AM2012-3825

Abstract 2528: Therapeutic targeting of a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by PCFT

Abstract The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, such that PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments (pH 6.2-6.8). PCFT transcripts and protein are high in human solid tumor tissues and cell lines (especially hepatic and ovarian tumors) and limited in most human normal tissues. We identified a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (AG71) that is a selective transport substrate for PCFT over the ubiquitously expressed reduced folate carrier (RFC) and a potent inhibitor of the de novo purine nucleotide biosynthetic enzyme glycinamide ribonucleotide (GAR) formyl transferase (GARFTase). Both AG71 and the clinically relevant antifolate pemetrexed (Pmx) were potently inhibitory toward proliferation of a HeLa subline (R1-11-PCFT4) that lacked RFC expression and was engineered to ectopically express physiologically relevant PCFT levels. Unlike Pmx, AG71 did not inhibit proliferation of R1-11-RFC6 cells which express physiologically relevant RFC levels but no PCFT. In colony-forming assays at pH 6.8, AG71 inhibited colony formation of R1-11-PCFT4 cells in a dose- and time-dependent manner, comparable to Pmx. We measured uptake of [3H]AG71 in R1-11-PCFT4 cells and found that transport was optimal at pH 5.5 but still significant at pH 6.8. At pH 6.8, AG71 was metabolized by folylpoly-γ-glutamate synthase to AG71 polyglutamates, as detected by HPLC. GARFTase was validated as a major intracellular target with an in situ GARFTase activity assay at pH 6.8 involving [14C]glycine incorporation into [14C]formyl GAR. In HepG2 human hepatocellular carcinoma cells, PCFT levels were elevated and [3H]AG71 was transported at pH 6.8, whereupon it was extensively metabolized to polyglutamates. By proliferation assays, AG71 was potently inhibitory (IC50 of 227.5 nM) toward HepG2 cells. To test in vivo efficacy of AG71, SCID mice were implanted with subcutaneous HepG2 tumors and treated with AG71 (180 mg/kg × 3d). Compound AG71 was highly active against HepG2 tumors. In conclusion, we have established the expression patterns and pH-specific transport for PCFT in human solid tumor cells and demonstrated that the novel PCFT-targeted antifolate AG71 can be internalized into tumor cells by PCFT leading to significant growth delay of HepG2 xenografts. Our findings offer exciting new therapeutic possibilities for selectively targeting chemotherapy to tumors with novel antifolates based on their acidic microenvironments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2528. doi:10.1158/1538-7445.AM2011-2528

Molecular cloning and tissue distribution of reduced folate carrier and effect of dietary folate supplementation on the expression of reduced folate carrier in laying hens

The reduced folate carrier (RFC) has been postulated to be a major entity for folate transport activity in humans and other mammals. However, there are limited reports of the importance of RFC in an avian system. In the current study, therefore, the molecular cloning and tissue distribution of RFC, as well as the effect of dietary folate supplementation on the expression of this transporter, were investigated in the chicken. Shaver White laying hens (n = 8 per diet) received 3 wheat-based diets containing the following: 1) no supplemental folate, 2) folic acid (10.00 mg/kg), or 3) 5-methyltetrahydrofolate (11.30 mg/kg) for 21 d. The mRNA expression levels were analyzed by quantitative real-time PCR. The results showed that the cloned partial RFC cDNA containing the full coding region from duodenum was 99% homologous to the reference gene available in GenBank. A broad expression profile of RFC transcripts was observed, with RFC mRNA detected in the brain, liver, kidney, spleen, lung, intestine, ovary, and testis, as well as other tissues. Real-time PCR analysis revealed that no significant differences (P > 0.05) due to diet were found in the mRNA levels of RFC in the duodenum and cecum. However, compared with the basal diet, jejunal mRNA levels of RFC were decreased (P < 0.05) in hens fed with the 5-methyltetrahydrofolate diet, but the reduction did not reach significance (P = 0.077) in the hens fed the folic acid diet. Overall, the current study demonstrated that the RFC cDNA containing the full coding region was successfully cloned from the duodenum of laying hens. The wide tissue distribution of RFC transcripts is suggestive of an important role of RFC in the process of folate transport in the chicken. Moreover, dietary folate supplementation could downregulate the jejunal mRNA expression of RFC. Such findings will lay the foundation of future work involving the RFC in avian systems, including laying hens.

Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier.

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; Kt, approximately 45 nmol/L) than methotrexate (MTX; Kt, approximately 1 micromol/L) with comparable Vmax values. The Ki values for folic acid, ZD9331, and ZD1694 were approximately 400-600 nmol/L, and the Ki values for PT523, PT632, and trimetrexate were >50 micromol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 micromol/L), but Vmax was decreased 4.5-fold. There was no difference in the Kt for PMX (approximately 1.2 micromol/L) or the Ki for folic acid (approximately 130 micromol/L) or PT523 ( approximately 0.2 micromol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl- (HepG2-B > R5), Na+ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

Reduced folate carrier protein expression in osteosarcoma

High-dose methotrexate (MTX) is an important component of current protocols for the treatment of osteosarcoma. Although MTX uptake proceeds primarily through the reduced folate carrier (RFC) protein and efflux occurs via multidrug resistance protein 1 (MRP1), RFC protein expression in osteosarcoma remains unexamined. RFC and MRP1 expression (normalized to beta-actin expression) was examined with Western blot analysis in 11 osteosarcoma specimens obtained at diagnosis and 9 osteosarcoma specimens obtained on recurrence. The average RFC level in specimens obtained on recurrence was significantly higher than the level in specimens obtained at diagnosis (P = 0.0005). Furthermore, in all three matched pairs of diagnosis and recurrence specimens, RFC levels were higher in recurrence specimens than in the corresponding diagnosis specimens. Potential correlations between RFC and MRP1 expression and histologic response to preoperative chemotherapy were examined. Tumors with poor histologic responses (i.e., </= 90% necrosis) had significantly lower RFC levels than did those with favorable responses to chemotherapy (P = 0.0016). In contrast, there was no correlation between MRP1 levels at diagnosis and histologic response to chemotherapy (P = 0.8764). The elevated MRP1 levels in specimens obtained on recurrence relative to MRP1 levels in specimens obtained at diagnosis were not statistically significant (P = 0.2056). The significant correlation between low RFC levels at diagnosis and poor histologic response to preoperative chemotherapy suggests that RFC levels at diagnosis may be a useful predictor of chemosensitivity and warrants large-scale studies. In addition, postchemotherapy progression to recurrence is associated with a significant increase in RFC expression. To our knowledge, the current study is the first to examine RFC protein levels in tumor specimens. Cancer 2003.

Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates.

Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26-A, C26-10, C26-G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5-fluorouracil (5-FU) alone or in combination with leucovorin (LV) (2-5-fold), to the thymidylate synthase (TS) inhibitors, AG337 (2-7-fold), ZD1694 (3-49-fold), ZD9331 (3-40-fold), LY231514 (2-21-fold) or GW1843U89 (4-29-fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2-50-fold) compared with their HF variants cultured in standard medium containing up to 8 microM folic acid. LV could only increase sensitivity to 5-FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2-7-fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7-fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3-fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5-FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5-FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models.

Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A Mechanism for Ploidy but not Lineage Differences in Methotrexate Accumulation

AbstractMethotrexate (MTX) is one of the most active and widely used agents for the treatment of acute lymphoblastic leukemia (ALL). To elucidate the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of the reduced folate carrier (RFC) was assessed by reverse transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a 60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with nonhyperdiploid ALL (median, 2.1; P < .0006), but no significant difference between nonhyperdiploid B-lineage and T-lineage ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21) were significantly related to chromosome 21 copy number (P = .0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene copy number, MTX-PG accumulation was compared in ALL blasts isolated from 121 patients treated with either low-dose MTX (LDMTX; n = 60) or high-dose MTX (HDMTX; n = 61). After LDMTX, MTX-PG accumulation was highest in hyperdiploid B-lineage ALL with 4 copies of chromosome 21 (P = .011), but MTX-PG accumulation was not significantly related to chromosome 21 copy number after HDMTX (P = .24). These data show higher RFC expression as a mechanism for greater MTX accumulation in hyperdiploid B-lineage ALL and indicate that lineage differences in MTX-PG accumulation are not due to lower RFC expression in T-lineage ALL.

Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A Mechanism for Ploidy but not Lineage Differences in Methotrexate Accumulation

Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute lymphoblastic leukemia (ALL). To elucidate the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of the reduced folate carrier (RFC) was assessed by reverse transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a 60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with nonhyperdiploid ALL (median, 2.1; P &lt; .0006), but no significant difference between nonhyperdiploid B-lineage and T-lineage ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21) were significantly related to chromosome 21 copy number (P = .0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene copy number, MTX-PG accumulation was compared in ALL blasts isolated from 121 patients treated with either low-dose MTX (LDMTX; n = 60) or high-dose MTX (HDMTX; n = 61). After LDMTX, MTX-PG accumulation was highest in hyperdiploid B-lineage ALL with 4 copies of chromosome 21 (P = .011), but MTX-PG accumulation was not significantly related to chromosome 21 copy number after HDMTX (P = .24). These data show higher RFC expression as a mechanism for greater MTX accumulation in hyperdiploid B-lineage ALL and indicate that lineage differences in MTX-PG accumulation are not due to lower RFC expression in T-lineage ALL.

Role of folate receptor and reduced folate carrier in the transport of 5-methyltetrahydrofolic acid in human ovarian carcinoma cells.

Folate receptor-alpha (FR-alpha) is generally over-expressed in non-mucinous human ovarian carcinomas. The meaning of FR-alpha over-expression and its role in the 5-methyltetrahydrofolic acid (N5-CH3-H4PteGlu) transport in such tumors is not clear, especially compared with the reduced folate carrier (RFC), the other known folate transporter. In this study, we analyzed molecular FR-alpha and RFC expression in 16 ovarian carcinoma tissues and in 5 ovarian carcinoma cell lines using competitive PCR. Co-expression of the 2 transporters was found both in vivo and in vitro. FR-alpha mRNA expression in the cell lines was in good agreement with the corresponding protein expression evaluated by cellular folic acid binding and immunofluorescence analysis, using a specific monoclonal antibody (MAb) (MOv18). Moreover, RFC mRNA expression levels were consistent with the selective cellular binding of N-hydroxysuccinimide of [3H]-methotrexate (NHS-MTX). The 5 ovarian carcinoma cell lines (IGROV-1, SW-626, SKOV-3, OVCAR-3 and OAW-42), grown at physiological N5-CH3-H4PteGlu concentrations (20 nM) and expressing FR-alpha and RFC levels superimposable to those observed in vivo, were used as in vitro cellular model to evaluate the different contribution of FR-alpha and RFC to the transport of N5-CH3-H4PteGlu. The cytoplasmic N5-CH3-[3H]H4PteGlu accumulation observed in each cell line was approximately linear over 4 hr of incubation, but there was no correlation between the rate of folate internalization and FR-alpha and RFC expression levels. Furthermore, the selective inhibition of FR-alpha and RFC functionality allowed us to distinguish their differential role on the overall N5-CH3-[3H]H4PteGlu intracellular delivery. Treatment with the N-hydroxysuccinimide of folic acid, which blocks FR-alpha activity, showed only a partial inhibition (about 20%) of folate internalization in all the cell lines. In contrast, the inhibition of RFC by NHS-MTX, under conditions that did not affect FR-alpha functionality, generally reduced folate accumulation by more than 70%. Only one cell line (IGROV-1) showed a comparable contribution of the 2 transport systems. Our findings suggest that in ovarian carcinomas, in spite of its over-expression, FR-alpha generally plays a minor role in N5-CH3-H4PteGlu transport compared with RFC.