5-methyltetrahydrofolate and Colonic-Neoplasms

The purpose of this study was to test the hypothesis that 5-methyltetrahydrofolate (Me-THF), a source of reduced folates alternative to leucovorin, could effectively modulate 5-fluorouracil's (5-FU) cytotoxic activity in patients with advanced colon cancer. A total of 23 patients were enrolled in a phase 11 trial; they received 5-FU as a 30-min infusion at a dose of 370 mg/m2 following a rapid i.v. push of 200 mg/m2 Me-THF, both drugs being given for 5 consecutive days. Cycles were repeated every 4 weeks until disease progression. No patient achieved a complete response. In all, 4 patients showed a partial response (17.4%), 7 developed stable disease (30.4%), and the remaining 12 (52.2%) progressed. Toxicity was acceptable and never exceeded WHO grade III intensity. According to our experience, the MeTHF/5-FU combination does not appear to be an effective treatment for advanced colon cancer. Despite its low toxic profile, in our opinion its wider use should be discouraged.

Topics: Adult; Aged; Antidotes; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Death; Colonic Neoplasms; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Neoplasm Metastasis; Tetrahydrofolates

Topics: Adenocarcinoma; Adult; Aged; Clinical Trials as Topic; Colonic Neoplasms; Drug Therapy, Combination; Female; Fluorouracil; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Pelvic Neoplasms; Rectal Neoplasms; Tetrahydrofolates

In this study, 50 patients were randomly assigned to treatment with 5-fluorouracil (FUra) or FUra plus high-dose continuous-infusion folinic acid. Five of 27 evaluable patients in the FUra group versus 10 of 21 patients in the FUra plus folinic acid arm of the study had objective partial remissions, P = 0.02. Time to progression was 3.9 months for FUra and 8.0 months for FUra and folinic acid, P = 0.006; however, median survivals (11.9 versus 14.5 months) were not different in this crossover study. Toxicity in both treatment arms was mild, although patients receiving FUra plus folinic acid experienced significantly more stomatitis than patients treated with FUra alone. This study suggests that high-dose, continuous-infusion folinic acid, which produces a steady-state level of biologically active folates of 10 microM, significantly increases the therapeutic activity of FUra.

Topics: Adenocarcinoma; Biological Availability; Colonic Neoplasms; Fluorouracil; Hematopoiesis; Humans; Infusions, Intravenous; Leucovorin; Rectal Neoplasms; Tetrahydrofolates

Folic acid (FA) consumption at high levels has been associated with colon cancer risk. Several mechanisms have been proposed to explain this association. The Notch signal pathway has been implicated in the regulation of cellular proliferation. Our aim was to demonstrate that high concentrations of FA or its reduced form, 5-methyltetrahydrofolic acid (5-MTHF), increase colorectal carcinoma HT29 cell proliferation through an increase of Notch1 activation and to prove if the inhibition of Notch1 activation by gamma secretase inhibitor, reduce the effect of folic acid. HT29 cells were cultured in high (400 nM), low (20 nM), or 0 nM FA or 5-MTHF concentrations during 96 h with or without DAPT (gamma secretase inhibitor). Cell proliferation was determined by the methylthiazole tetrazolium method, and Notch1-intracellular domain (NICD) was analyzed by flow cytometry. HT29 cells exposed to 400 nM FA or 5-MTHF showed higher proliferation rate than those exposed to 20 nM of FA or 5-MTHF (P < 0.01) during 96 h. NICD expression increased at higher FA or 5-MTHF concentrations compared with lower concentrations (P < 0.01). This effect on proliferation was partially reversible when we blocked Notch1 activation with the inhibitor of γ-secretase (P < 0.05).These data suggest that high concentration of FA and 5-MTHF induce HT29 cell proliferation activating Notch1 pathway.

Topics: Amyloid Precursor Protein Secretases; Cell Proliferation; Colonic Neoplasms; Folate Receptor 1; Folic Acid; HT29 Cells; Humans; Receptor, Notch1; Signal Transduction; Tetrahydrofolates

Although accumulating evidence suggests a chemopreventive role for folic acid (FA) in colorectal carcinogenesis, the underlying mechanisms are largely unknown. Previously, we reported that supplemental FA inhibits the expression and activation of epidermal growth factor receptor (EGFR) in colon cancer cell lines. To determine the mechanism(s) by which FA affects EGFR function, we have examined whether and to what extent supplemental FA or its metabolites 5-methyltetrahydrofolate (MTF), dihydrofolate (DF), and tetrahydrofolate (TF) will modulate basal and serum-induced activation of the EGFR promoter in the HCT-116 colon cancer cell line. HCT-116 cells were preincubated with or without (control) FA or one of its metabolites (10 microg/ml) for 48 h, transfected with the EGFR promoter luciferase reporter construct, and incubated for 48 h with FA, DF, TF, or 5-MTF in the absence or presence of 10% FBS. Supplemental FA as well as its metabolites markedly inhibited EGFR promoter activity and its methylation status. Exposure of the cells to 10% FBS caused a marked stimulation of EGFR promoter activity and its expression, both of which were greatly abrogated by supplemental FA and 5-MTF. In contrast, serum-induced activation of c-fos promoter activity was unaffected by 5-MTF. The 5-MTF-induced inhibition of serum-mediated stimulation of EGFR promoter activity and EGFR expression was reversed when methylation was inhibited by 5-aza-2'-deoxycytidine. Our data suggest that FA and its metabolite 5-MTF inhibit EGFR promoter activity in colon cancer cells by enhancing methylation. This could partly be responsible for FA-mediated inhibition of growth-related processes in colorectal neoplasia.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Blotting, Western; Cell Line, Tumor; Cells, Cultured; Colonic Neoplasms; Culture Media, Serum-Free; Decitabine; DNA Methylation; DNA Primers; ErbB Receptors; Folic Acid; Humans; Luciferases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Tetrahydrofolates; Transfection

There is an increasing evidence, stemming from epidemiological studies as well as studies performed in human biopsies and animal and cell culture models, suggesting that folate is chemopreventive in colonic carcinogenesis. Hyperhomocysteinemia is frequently associated with folate deficiency. Homocysteine, an amino acid, is metabolized to methionine in a 5-methyltetrahydrofolate (5-MTHF) dependent reaction.. The aim of this study was i) to evaluate the effects of folate and its metabolites on growth and cell cycle progression in human colon cancer cells (Caco-2) in culture, and ii) to assess the effects of exogenous homocysteine on colon cancer cell proliferation. iii) Having found that homocysteine enhances colon cancer cell growth while metabolites of folate inhibit cell proliferation, we investigated the effects of simultaneous treatment in colon cancer cells.. Caco-2 cells were incubated either with homocysteine (0.1-10 microM), and/or with folic acid and its metabolites (0.625-10 microg/ml). Cell proliferation was determined after 24 h and 48 h by measuring 5- bromo-2'-desoxyuridine (BrdU) incorporation. Additionally, the cells were trypsinized and prepared for cell cycle determination using propidium iodide for DNA staining. The stained cells were analyzed using a flow cytometer.. Folate inhibited cell proliferation moderately within 24 h. Its metabolites, dihydrofolate and 5-MTHF were more potent inhibitors of cell growth. Treatment with folate and 5-MTHF resulted in the accumulation of cells in G1-G0 phase of the cell cycle and decreased the number of cells in G2-M phase. In addition, cells treated with 5-MTHF were predominantly accumulated in the S-phase. There was no difference in cell cycle progression of Caco-2 cells treated with homocysteine in comparison to controls. In homocysteine-treated cells, both folate and 5-MTHF reversed the homocysteine-induced enhancement of growth. In contrast, folate reduced the Caco-2 cell growth rate to control values and 5-MTHF depleted growth of homocysteine-treated cells to levels significantly lower than controls.. Our data suggest that 5-MTHF, being the key metabolite in both the folate and homocysteine metabolic pathway, is the main modulator of growth-promoting actions of homocysteine as well as antiproliferative effects of folate in colon cancer cells.

Topics: Caco-2 Cells; Cell Cycle; Cell Death; Cell Division; Colonic Neoplasms; Flow Cytometry; Folic Acid; Hematinics; Homocysteine; Humans; In Vitro Techniques; Tetrahydrofolates; Time Factors

Topics: Adenocarcinoma; Animals; Caco-2 Cells; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Disease Models, Animal; DNA Methylation; Folic Acid; Humans; Tetrahydrofolates

The therapeutic activity of FUra alone or combined with [6RS]LV doses ranging from 50 to 1,000 mg/m2 was examined in eight colon adenocarcinoma xenografts, of which five were established from adult neoplasms (HxELC2, HxGC3, HxVRC5, HxHC1, and HxGC3/c1TK-c3 selected for TK deficiency) and three were derived from adolescent tumors (HxSJC3A, HxSJC3B, and HxSJC2). The growth-inhibitory effects of FUra were potentiated by higher doses of [6RS]LV (500-1,000 mg/m2) in three lines (HxGC3/c1TK-c3, HxSJC3A, and HxSJC3B) and by a low dose of [6RS]LV in only one tumor (HxVRC5). Expansion of pools of CH2-H4PteGlun+H4PteGlun (greater than or equal to 2.4-fold) in response to higher doses of [6RS]LV was obtained in all lines except HxHC1. Metabolism of [6RS]LV was high in HxVRC5, with high levels of 5-CH3-H4PteGlu being detected, but not in HxHC1, in which levels of 5-CH3-H4PteGlu and CH = H4PteGlu+10-CHO-H4PteGlu remained relatively low. In the adolescent tumors, levels of CH = H4PteGlu+10-CHO-H4PteGlu were consistently higher than those of 5-CH3-H4PteGlu following [6RS]LV administration, and in HxSJC3A, in which pools of CH2-H4PteGlun+H4PteGlun were significantly expanded, 5-CH3-H4PteGlu concentrations were lower than those observed in the other two lines. The sensitivity of tumors to FUra +/- [6RS]LV and the characteristics of [6S]LV metabolism did not correlate with the activity of CH = H4PteGlu synthetase, the enzyme responsible for the initial cellular metabolism of [6S]LV to CH = H4PteGlu. Thus, no single metabolic phenotype correlated with the [6RS]LV-induced expansion of CH2-H4PteGlun+H4PteGlun pools. Potentiation of the therapeutic efficacy of FUra by [6RS]LV was observed in HxGC3/c1TK-c3 xenografts but not in parent HxGC3 tumors, demonstrating the influence of dThd salvage capability in the response to FUra-[6RS]LV combinations. Plasma dThd concentrations in CBA/CaJ mice were high (1.1 microM). The present data therefore demonstrate the importance of (1) higher doses of [6RS]LV, (2) expansion of pools of CH2-H4PteGlun+H4PteGlun, and (3) dThd salvage capability in potentiation of the therapeutic efficacy of FUra in colon adenocarcinoma xenografts. The plasma levels of FUra achieved in mice are presented.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Colonic Neoplasms; Fluorouracil; Humans; Leucovorin; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Tetrahydrofolates

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H4PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), and the distribution of their polyglutamate species have been examined. Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m2) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H4-PteGlu and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t1/2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC2 and HxGC3 tumors, pools of CH2-H4PteGlun and H4PteGlun were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH2-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin. In HxGC3 tumors, CH2-H4PteGlu4-5 were elevated more rapidly than in line HxELC2, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH2-H4PteGlu5 until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH2-H4PteGlu3-5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-3H]5-fluorodeoxyuridine monophosphate ([6-3H]FdUMP) to thymidylate synthase, and intratumor pools of CH2-H4PteGlun and H4PteGlun were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Floxuridine; Half-Life; Humans; Injections, Intravenous; Leucovorin; Mice; Mice, Inbred CBA; Tetrahydrofolates; Thymidylate Synthase; Time Factors

Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.

Topics: Adenocarcinoma; Animals; Biotransformation; Cell Line; Colonic Neoplasms; Female; Formyltetrahydrofolates; Humans; Kinetics; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Pteroylpolyglutamic Acids; Tetrahydrofolates; Transplantation, Heterologous

Plasma pharmacokinetics of high-dose (500 mg/m2) leucovorin calcium (dl-5-formyltetrahydrofolic acid [dl-CF]) and fluorouracil (FUra) have been evaluated in patients with advanced colorectal cancer treated with the combination of FUra and dl-CF by two different intravenous (IV) schedules: (A) In patients with no prior chemotherapy, dl-CF was administered by a two-hour IV infusion and FUra by rapid IV injection one hour after the start of the dl-CF infusion and (B) in previously treated patients, dl-CF and FUra were administered by five-day continuous IV infusion (CI). Following the two-hour infusion of dl-CF, mean peak plasma concentration and elimination half-life of I-5-formyltetrahydrofolic acid (I-CF) were 24 +/- 6 mumol/L and 0.8 +/- 0.1 hour, respectively. CI of dl-CF over five days yielded a mean steady-state plasma level of I-CF of only 1.2 +/- 0.5 mumol/L. Peak and steady-state plasma concentrations of the metabolite 5-methyl tetrahydrofolic acid were comparable in the two schedules (17 +/- 8 mumol/L for the two-hour infusion and 12 +/- 5 mumol/L for the CI). Areas under the concentration v time curve (AUC) of total reduced folates were significantly greater under conditions of CI: 89.0 v 16.7 mmol/L/min for the two-hour infusion. In tumor tissue, 5,10-methylenetetrahydrofolate increased eight-fold two to four hours following the two-hour infusion and two-fold during the CI of dl-CF and FUra. Inhibition of thymidylate synthase (dTMP-S) by the two-hour and CI infusion schedules were 66% v 39%, respectively. The observed differences in the intracellular dTMP-S folate cofactor pools and the degree of inhibition of dTMP-S achieved in patients treated by two different schedules may be due to differences in the biochemical properties and/or to differences in the modulation of FUra metabolism by folate of tumor tissues obtained from newly diagnosed and previously treated patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Chromatography, High Pressure Liquid; Colonic Neoplasms; Fluorouracil; Half-Life; Humans; Infusions, Intravenous; Injections, Intravenous; Leucovorin; Rectal Neoplasms; Tetrahydrofolates

Thirty-eight patients with advanced colorectal adenocarcinoma were treated with the following regimen: N5-10-methyltetrahydrofolate (MTHF) (200 mg/m2/day) and 5-fluorouracil (5-FU) (375 mg/m2/day) given concomitantly, consecutively for 5 days, every 4 weeks, in order to evaluate the potential advantage derived from the biochemical enhancement of cytotoxic activity of 5-FU by high-dose reduced folates. Of 33 evaluable patients (six of whom had received prior 5-FU chemotherapy) three untreated patients achieved a partial response (9.1%) lasting 84, 281 and 401 days; 24 patients (72.7%) had stable disease lasting a median of 150 days (range 60-304 days). The overall toxicity was acceptable: two patients had severe cardiac symptoms. Pharmacokinetics and biochemical studies seem necessary to determine the optimal dosage and timing of 5-FU and folates.

Topics: Adenocarcinoma; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Colonic Neoplasms; Drug Evaluation; Female; Fluorouracil; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Rectal Neoplasms; Tetrahydrofolates