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PubMed2004

Antifolates targeted specifically to the folate receptor.

Most antifolate drugs are efficiently transported by the reduced-folate carrier (RFC). However, several also bind with high affinity to the alpha-isoform of the folate receptor (alpha-FR) and there is evidence to suggest that this transport mechanism may contribute to their activity when the receptor is highly overexpressed or when the extracellular folate concentration is very low. In particular, the presence of the alpha-FR on tumour cell lines sensitises them to brief exposures to ZD9331. Nevertheless, it is the ubiquitous expression of the RFC in normal tissues that reduces patient tolerability to antifolate drugs. The overexpression of the alpha-FR in some epithelial tumours and its restricted distribution in normal tissues suggests an opportunity for the development of antifolates specifically targeted at alpha-FR overexpressing tumours. Potent cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) have been discovered with high and low affinity for the alpha-FR and RFC, respectively. This class of agent is represented by CB300638 (TS Ki=0.24 nM) that displays high potency (IC50 approximately 3 nM) for A431-FBP cells (transfected with the alpha-FR) and KB cells (constitutive overexpression). Importantly, this activity is approximately 300-fold higher than for alpha-FR negative cell lines such as A431. In mice bearing the KB tumour xenograft we have demonstrated localisation of CB300638 to tumour and, more importantly, specific inhibition of TS in tumour and not in normal tissues. Data supports the clinical development of this class of agent with the prediction that toxicity would be reduced compared with conventional antifolate drugs. There are a number of challenges to this development posed by the uniqueness of the compounds and these are discussed.

Animals↗

PubMed2004

Impaired CREB-1 phosphorylation in antifolate-resistant cell lines with down-regulation of the reduced folate carrier gene.

The human reduced folate carrier (hRFC) is the dominant transporter for the uptake of antifolates used in cancer chemotherapy. We have shown recently that decreased cAMP-responsive element (CRE)-dependent transcription contributes to the loss of hRFC gene expression in multiple antifolate-resistant cell lines. This was associated with markedly decreased levels of phosphorylated cAMP response element-binding protein 1 (pCREB-1) and CRE-binding. Consistent with the autoregulation of CREB-1 gene expression by pCREB-1, prominently decreased CREB-1 mRNA levels were observed in antifolate-resistant cells. We therefore explored the possibility that these cells were defective in CREB-1 phosphorylation, thereby resulting in down-regulation of some cAMP-responsive genes. Two-dimensional gel electrophoresis revealed that CREB-1 and its phosphoisoforms were markedly decreased in these cells. Treatment with forskolin, an activator of adenylyl cyclase, restored both CREB-1 and pCREB-1 levels; this resulted in the restoration of CRE-binding, CRE-reporter activity, and CREB-1 and RFC mRNA levels. Hence, the protein kinase A pathway was examined using various agents that augment intracellular cAMP levels, including cholera toxin, an upstream agonist that renders stimulatory G-proteins (Galphas) constitutively active. Treatment of antifolate-resistant cells with these agents resulted in the restoration of pCREB-1 levels and CRE-reporter activity. Furthermore, transient transfection with a constitutively transcriptionally active VP16-CREB-1 that does not require phosphorylation for its activity resulted in restoration of CREB mRNA levels but not pCREB-1 levels. This is the first demonstration that resistance to various antifolates may potentially be associated with impaired activity of Galphas or their coupled receptors, resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP-responsive genes.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Selective delivery of CB300638, a cyclopenta[g]quinazoline-based thymidylate synthase inhibitor into human tumor cell lines overexpressing the alpha-isoform of the folate receptor.

The alpha-isoform of the glycosylphosphatidylinositol cell membrane tethered folate receptor (alpha-FR) is overexpressed in some carcinomas (notably ovarian carcinomas) relative to normal tissues. The nonpolyglutamatable folate-based thymidylate synthase (TS) inhibitor, CB300638 (TS K(i) = 0.24 nM) displayed an IC(50) of 0.0028 microM for the inhibition of the growth of human A431-FBP cells transfected with the alpha-FR. In contrast, the IC(50) for the neotransfected A431 cells was 0.81 microM (300-fold higher). Similarly, this compound inhibited the growth of human KB cells that constitutively overexpress the alpha-FR with an IC(50) of 0.0036 microM. These data were derived from cells grown in a physiological concentration of folate (20 nM R,S-leucovorin). Incubation of KB cells with a 1 microM excess of folic acid (FA), to selectively block uptake via the alpha-FR, increased the CB300638 IC(50) to 0.39 microM. The relatively low potency of CB300638 under these conditions, or in cell lines not expressing the alpha-FR, is ascribed to its low affinity for the ubiquitously expressed folate transporter, the reduced-folate carrier (K(i) for inhibition of [(3)H]methotrexate transport >100 microM). The high potency of CB300638 in alpha-FR-overexpressing cell lines is attributable to high affinity of the alpha-FR (53% of FA) and efficient endosomal trafficking mediated by the alpha-FR. Sixteen-h exposure to CB300638 inhibited the rate of (3)H(2)O release from 5-[(3)H]dUrd (in situ TS assay) in A431, A431-FBP, and KB cells with IC(50) values of 0.1 microM, 0.005 microM, and 0.002 microM, respectively. The coaddition of 1 micro M FA increased the IC(50)s for A431-FBP and KB cells to approximately 0.1 microM consistent with alpha-FR-mediated transport of CB300638. In conclusion, alpha-FR-mediated uptake of CB300638 leads to TS and growth inhibition that is highly selective for alpha-FR overexpressing tumor cell lines. The low expression of the alpha-FR in normal tissues, particularly those sensitive to TS inhibitors, together with the low affinity of CB300638 for the reduced-folate carrier, suggests that the compound may have potential as an antitumor agent with a high therapeutic index.

Antineoplastic Agents↗

PubMed1999

Inhibitory effects of prostaglandin A1 on membrane transport of folates mediated by both the reduced folate carrier and ATP-driven exporters.

Studies are reported that describe the multifaceted inhibitory effects of prostaglandin A1 (PGA1) on processes that govern the transport of folates across the plasma membrane of Chinese hamster ovary (CHO) cells: the reduced folate carrier, RFC1, and ATP-dependent exporters. PGA1 was a noncompetitive inhibitor of MTX influx mediated by RFC1 with a Ki of approximately 21 microM. The onset of inhibition was virtually instantaneous, not reversible, and appeared to require the incorporation of PGA1 into the lipid membrane; surface adsorption alone was insufficient for inhibition of RFC1 transport activity. In contrast, the effect of PGA1 on folic acid transport was small (approximately 20% inhibition of total influx), consistent with the observation that the major portion of folic acid transport in CHO cells is mediated by a low pH mechanism distinct from RFC1. PGA1 was also a potent inhibitor of the ATP-driven efflux of both MTX and folic acid. At a concentration of 7 microM PGA1, the efflux rate constants for these folates were depressed by approximately 70 and approximately 50%, respectively. The net effects of PGA1 on the bidirectional folate fluxes translated into marked alterations in net transport. The addition of 7 microM PGA1 to cells at steady state with 1 microM MTX produced a rapid onset of net uptake and the achievement of an approximately 3-fold increase in the steady-state free MTX level as compared with untreated CHO cells. The addition of 7 microM PGA1 to cells at steady state with 1 microM folic acid produced an approximately 5-fold increase in the free folate level. These studies establish PGA1 as a potent inhibitor of both the reduced folate carrier and ATP-driven folate exporter(s). The noncompetitive nature of the inhibition of RFC1 is unique among anionic compounds, which are usually competitive inhibitors of the carrier.

Adenosine Triphosphate↗

PubMed1998

A reduced folate carrier mutation produces substrate-dependent alterations in carrier mobility in murine leukemia cells and methotrexate resistance with conservation of growth in 5-formyltetrahydrofolate.

With 5-formyltetrahydrofolate (5-CHO-THF) as the folate source a methotrexate (MTX) transport-deficient murine leukemia cell line, L1210-G1a, was isolated after chemical mutagenesis and MTX selection. This cell line was 10-fold resistant to MTX in comparison to parental L1210 cells, yet the EC50 for 5-CHO-THF was increased by a factor of only 2. The initial uptake of MTX, at a concentration of 1 microM, was decreased by a factor of 40, whereas influx of 5-CHO-THF dropped by a factor of only 8. This difference in initial uptake rates was attributed solely to changes in influx Vmax without a significant change in Km. Whereas the RFC1 mRNA level in L1210-G1a cells was indistinguishable from that of parental L1210 cells, a serine to asparagine substitution was identified at amino acid 46 within the first predicted transmembrane domain. This was a result of a homozygous mutation of G-->A in the genome. Transfection of the mutated RFC1 cDNA into MTXrA cells, which lack functional endogenous carrier, resulted in a clonal derivative MTXrA-S46N. The increase in influx of 5-CHO-THF and 5-CH3THF was 5 and 13 times greater than that for MTX in the transfectant, consistent with the influx ratio in the L1210-G1a line. The functional expression of the mutated RFC1 reduced the growth requirement for 5-CHO-THF by a factor of 30, compared with only a 3-fold decrease in the MTX IC50. This represents the first reported RFC1 mutation that confers resistance to MTX due to a markedly impaired influx with relative conservation of reduced folate transport. The kinetic changes are consistent with a substrate-dependent alteration in carrier mobility that favors reduced folates over MTX. These changes may account for the development of MTX resistance due to impaired drug transport in vivo, allowing tumor cells to meet their folate requirement with 5-CH3THF, the predominant blood folate.

Animals↗

PubMed2002

Synthesis and evaluation of 99mTc(CO)(3)-DTPA-folate as a folate-receptor-targeted radiopharmaceutical.

A folate-receptor-targeted 99mTc-radiopharmaceutical, [99mTc]Tc(CO)(3)DTPA-folate, was prepared by heating [99mTc]Tc(CO)(3)(H(2)O)(3)(+) in an aqueous solution of the previously reported DTPA-folate conjugate. The radiotracer was HPLC purified (> 98% radiochemical purity) and evaluated in vitro and in vivo as an agent for targeting folate-receptor-positive cells. [99mTc]Tc(CO)(3)DTPA-folate experienced high, folate-receptor-specific uptake in human KB tumor cells. Intravenous administration of [99mTc]Tc(CO)(3)DTPA-folate to athymic mice bearing KB cell tumor xenografts resulted in 99mTc tumor uptake of 1.8 +/- 0.5 and 3.3 +/- 0.2%ID/g (n = 3) at 30 minutes and 4 hours post-injection, respectively. Tumor uptake was reduced when folic acid was co-administered with the intravenous [99mTc]Tc(CO)(3)DTPA-folate, consistent with radiopharmaceutical localization being mediated by the folate receptor.

Animals↗

PubMed1990

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization.

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.

Animals↗

PubMed1991

Conversion of an apparent 100 kDa folate binding protein from human milk to a 25 kDa molecular species by phospholipase D.

Gel filtration studies in the presence of Triton X-100 showed that treatment with phospholipase D reduced the apparent molecular size of the 100 kDa folate binding protein from human milk to 25 kDa, which is the molecular size of the low molecular weight folate binding protein. A phospholipase D induced cleavage of a hydrophobic phospholipid domain inserting the protein into Triton X-100 micelles could account for this phenomenon.

Carrier Proteins↗

PubMed1999

Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis.

OBJECTIVE: To investigate the expression of folate receptors (FR) and reduced folate carrier (RFC) and determine their relevance to methotrexate (MTX) transport in synovial mononuclear cells (SMC) from patients with rheumatoid arthritis (RA). METHODS: Levels of FR and RFC messenger RNA (mRNA) were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in SMC from RA patients and peripheral blood mononuclear cells from healthy donors. Expression of FR-beta mRNA and protein was determined by Northern blot and Western blot analyses in RA SMC and monocyte/macrophage-lineage cells. FR-beta expression and folic acid binding capacity on the cell surface were examined by flow cytometric analysis and 3H-folic acid binding analysis. Studies of the inhibition of 3H-MTX uptake in the presence of unlabeled folic acid were performed to investigate the uptake of MTX through FR in RA SMC. RESULTS: RT-PCR, Northern blot, and Western blot analyses showed that FR-beta mRNA and protein were expressed selectively in activated monocytes and CD14+ RA SMC. These cells exhibited folic acid binding capacity. Furthermore, the FR-beta protein was shown to have folic acid binding capacity. Uptake of 3H-MTX through RA SMC was significantly inhibited in the presence of unlabeled folic acid. CONCLUSION: These results demonstrate that FR-beta expression is selectively elevated in RA synovial macrophages and suggest that MTX is transported through FR-beta in RA synovial macrophages. The findings suggest that folate antagonists with higher affinity for FR-beta would be useful in the treatment of RA.

Arthritis, Rheumatoid↗

PubMed2006

Maternal MTR genotype contributes to the risk of non-syndromic cleft lip and palate in the Polish population.

The aetiology of non-syndromic cleft lip with or without cleft palate (CL/P) is very complex. It has been shown that polymorphic variants of genes encoding key proteins of folate and methionine metabolism might be important maternal risk factors of having a child with this craniofacial anomaly. Therefore, in our study, mothers with CL/P children as well as control mothers were examined for prevalence of polymorphisms of genes that encode methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase (MTHFD1) and reduced folate carrier 1 (RFC1). We observed that there were no statistical differences in allele and genotype frequencies of MTHFR c.677C>T, MTHFD1 c.1958G>A and RFC1 c.80G>A between mothers who had children with CL/P and control mothers. However, mothers with MTR c.2756AG or GG genotype displayed a 2.195-fold increased risk of having a child with CL/P (95% CI 1.189-4.050, p = 0.011). The mechanism by which polymorphic transition of MTR gene might increase the maternal risk of having CL/P progeny is unknown. Our observations are consistent with a significant role of the methyl cycle in the development of craniofacial structures in humans.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line.

Ohnuma et al reported a series of methotrexate-resistant MOLT-3 human T-cell acute lymphoblastic leukemia cell lines that showed decreasing methotrexate (MTX) uptake as the sublines acquired increasing MTX resistance (Cancer Res 45:1815, 1985). The alteration of MTX uptake kinetics in these cells, the intermediately resistant MOLT-3/MTX200 and the highly resistant MOLT-3/MTX10,000 cell lines, was attributed to a change in Vmax for methotrexate transport, without an apparent change in affinity of the transporter for MTX. We studied these cell lines to determine whether alteration of transcription or translation of the recently isolated reduced folate carrier gene (RFC1) was the cause of MTX transport deficiency in these cell lines. Reconstitution of RFC activity in MOLT-3/MTX10,000 cells by transduction with a murine RFC retroviral vector reversed MTX resistance and trimetrexate sensitivity. Although RFC1 RNA levels were unchanged in the resistant cell lines, FACS analysis using a polyclonal anti-RFC1 antibody showed no detectable RFC1 protein in the MOLT-3/MTX10,000 cells. Determination of the nucleotide sequence of RFC1 genes from MOLT-3/MTX10,000 cells revealed that this cell line contained 3 RFC1 alleles: a wild-type allele, an allele containing the premature stop codon at codon 40 and a third allele containing another mutation, which resulted in a premature stop codon at codon 25. We examined the relative expression of these alleles by determining the nucleotide sequence of 24 RFC1 cDNA subclones from MOLT-3/MTX10,000 cells and found that only one-third of these clones contained the wild-type sequence. Determination of the genomic sequence of RFC1 in MOLT-3/ MTX200 cells demonstrated that these cells were heterozygous for a mutation at codon 40, but were homozygous for the wild-type sequence at codon 25. Thus, the acquisition of MTX transport-deficiency in MOLT-3/MTX10,000 cells results from inactivating mutations of RFC1 gene alleles.

Alleles↗

PubMed1981

Evidence for a folic acid binding protein in human cell membrane.

Cell membranes were prepared by sucrose discontinuous gradient from human liver and human peripheral leukocytes and erythrocytes and from circulating leukocytes from patients with chronic granulocytic leukemia (CGL) and acute myeloblastic leukemia (AML). The membrane preparations from liver and from leukemic leukocytes were shown to bind tritiated folic acid. The membranes from normal leukocytes and erythrocytes did not show this binding capacity. The membrane preparation from liver and CGL leukocytes showed two peaks of binding eluting with proteins from Sephadex G-200. However, protein extracts of these membrane preparations showed only a single peak for labelled folic acid, eluting near but just after albumin. The binding capacity of the membranes for folic acid was partially inhibited by reduced folate analogues. It is concluded that the liver plasma cell membrane and the membranes of myeloblasts in AML and circulating leukocytes in CGL contain a binding protein for folic acid which may be concerned in the transport of folates into these cells.

Animals↗

PubMed1997

Folate-based thymidylate synthase inhibitors in cancer chemotherapy.

Understanding the relationship between chemical structure and biological properties of folate analogs, particularly their interactions with the target enzymes, transport proteins and folate-metabolizing enzyme, folylpolyglutamate synthetase (FPGS), has enabled the rational design and development of the selective thymidylate synthase (TS) inhibitors with folate-based structures for clinical uses. These compounds specifically inhibit TS devoid of concomitant effects at other loci, unlike 5-fluorouracil (5-FU). ZD1694 ('Tomudex') was designed as a non-nephrotoxic and highly active analog of N10-propargyl-5,8-dideazafolic acid (CB3717), which is a potent TS inhibitor but had unacceptable nephrotoxicity caused by its poor water solubility. The potent cytotoxic activity of ZD1694 is dependent upon active uptake into cells via the reduced folate carrier (RFC), and subsequent rapid and extensive metabolism to polyglutamate forms inside cells. Marked enhancement of the TS inhibitory activity has been noted as the glutamate chain is elongated. Polyglutamation is critical to the biological activity of ZD1694 against tumor and normal proliferating tissues. The retentive property of ZD1694 polyglutamates inside cells led to a single, infrequent administration schedule in clinical studies. ZD1694 has completed phase I and phase II evaluation with activity observed in several tumor types, particularly in colorectal cancer with a 26% objective response rate. A recent European phase III study of ZD1694, randomized against a 5-FU plus leucovorin regimen, demonstrated an equivalent response rate for advanced colorectal cancer (complete or partial responses; 20 versus 17%) and less toxicity than seen with the latter regimen. The newer selective TS inhibitors, which retain potency for TS inhibition but are not substrates for RFC and/or FPGS, are currently under clinical evaluation. These classes of compound may have benefits for circumvention of resistance by virtue of alterations in these protein functions and for the management of toxicity.

Animals↗

PubMedDate not supplied

DNA vaccination against the ovarian carcinoma-associated antigen folate receptor alpha (FRalpha) induces cytotoxic T lymphocyte and antibody responses in mice.

Human folate receptor alpha (FRalpha) is a folate-binding protein that is selectively overexpressed in ovarian carcinoma and has been regarded as a suitable target antigen for immunotherapy purposes. To study the possible use of this antigen in DNA vaccination, FRalpha cDNA was ligated into the VR1012 (Vical) expression vector under the transcriptional control of the cytomegalovirus promoter. A total of 100 microg of purified plasmid DNA was injected intramuscularly in BALB/c mice three times at 14-day intervals. At 10 days after the second injection, the sera of the animals (100%) displayed significant antibody titers (by indirect immunofluorescence and fluorescence-activated cell sorter analysis) against syngeneic C26 cells transduced with FRalpha, but not against unmodified C26 cells. Immunoglobulin G2a was the predominant isotype. In addition, specific cytotoxic T lymphocyte activity against FRalpha-transduced C26 cells could be detected in splenocytes from all immunized animals. Coinjection of a plasmid containing interleukin-2 cDNA increased both antibody titers and cytotoxic T lymphocyte activity. Challenge by subcutaneous injection with FRalpha-transduced C26 cells (performed 10 days after the third injection) showed a statistically significant delay in tumor growth. Vaccination with the FRalpha and interleukin-2 cDNA mixture, which was performed after an intravenous injection of FRalpha-transduced cells, enhanced the mean survival time and reduced the number of lung metastases, thus suggesting that such vaccination is effective even against preexisting tumor cells.

Animals↗

PubMed2004

Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate.

For folate-receptor-targeted anti-cancer therapy, doxorubicin aggregates in a nano-scale size were produced employing doxorubicin-polyethylene glycol-folate (DOX-PEG-FOL) conjugate. Doxorubicin and folate were respectively conjugated to alpha- and omega-terminal end group of a PEG chain. The conjugates assisted to form doxorubicin nano-aggregates with an average size of 200 nm in diameter when combined with an excess amount of deprotonated doxorubicin in an aqueous phase. Hydrophobically deprotonated doxorubicin molecules were aggregated within the core, while the DOX-PEG-FOL conjugates stabilized the aggregates with exposing folate moieties on the surface. The doxorubicin nano-aggregates showed a greater extent of intracellular uptake against folate-receptor-positive cancer cells than folate-receptor-negative cells, indicating that the cellular uptake occurred via a folate-receptor-mediated endocytosis mechanism. They also exhibited more potent cytotoxic effect on KB cells than free doxorubicin. In a human tumor xenograft nude mouse model, folate-targeted doxorubicin nano-aggregates significantly reduced the tumor volume compared to non-targeted doxorubicin aggregates or free doxorubicin. These results suggested that folate-targeted doxorubicin nano-aggregates could be a potentially useful delivery system for folate-receptor-positive cancer cells.

Animals↗

PubMed1995

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Animals↗

PubMed2000

Down-regulation of reduced folate carrier gene (RFC1) expression after exposure to methotrexate in ZR-75-1 breast cancer cells.

Methotrexate (MTX) is administered in intervals of one week or longer in the treatment of cancer and autoimmune disease. Early studies suggested that daily MTX administration was associated with decreased effectiveness and increased toxicity, leading to schedules of administration that include periodic intervals of rest during chronic MTX therapy. We hypothesized that these observations may be the result of the down-regulation of the reduced folate carrier, the major route of cellular uptake of both MTX and the endogenous folates, after MTX exposure. We exposed folate-depleted ZR-75-1 breast cancer cells to low-dose MTX in the presence of hypoxanthine, adenosine and thymidine. After 72 h, the initial rate of MTX uptake had decreased to 22% of the Day 0 value. Western blot analysis showed down-regulation of RFC1 protein expression, and Northern blot analysis showed a corresponding decrease in RFC1 RNA levels. Using an RT-PCR assay, we found that levels of RNA transcripts containing each of the three RFC1 5' noncoding exons were decreased after exposure to MTX, suggesting that MTX exposure causes transcriptional down-regulation of RFC1. Promoter-reporter construct assays demonstrated decreased activity of RFC1 promoter elements upstream of these exons after MTX exposure. Preexposure of the ZR-75-1 cells to 5-azacytidine, a DNA methylation inhibitor, further decreased MTX uptake rather than reverse the inhibition of RFC1 activity, indicating that RFC1 down-regulation after MTX exposure is not the result of methylation of the RFC1 promoter. In summary, these studies demonstrate that MTX exposure can down-regulate RFC1 expression and activity. These acute, inducible, epigenetic changes in RFC1 expression may ultimately be molded into the more permanent genetic changes that result in the transport-mediated MTX resistance that have been observed in MTX-resistant cell lines.

Antimetabolites, Antineoplastic↗

PubMed1997

Fostriecin: a review of the preclinical data.

Fostriecin is a novel antitumor antibiotic. In vitro studies showed that fostriecin inhibits DNA topoisomerase II (Topo II) catalytic activity, protein phosphatases involved with cell-cycle control and histone phosphatases. The relative contribution of these mechanisms to the antitumor activity has not been elucidated, but Topo II inhibition seems to be the major mechanism of action at in vitro cytotoxic fostriecin levels. Tumor cell lines with decreased Topo II content showed similar or increased sensitivity to fostriecin, compared to the parent cell lines. The reduced-folate carrier is probably responsible for the cellular uptake of fostriecin. The possible clinical consequences of these in vitro observations are discussed.

Alkenes↗