Affiliations of authors: Department of Medicine (KJS, YIK), Department of Nutritional Sciences (YIK), University of Toronto, Toronto, Ontario, Canada; Clinical Epidemiology Unit, Sunnybrook and Women's College Health Sciences Center, Toronto (RC); Academic Unit of Paediatrics and Obstetrics and Gynaecology, University of Leeds, West Yorkshire, U.K. (ZY); Department of Human Nutrition, School of Applied Sciences, Ourimbah Campus, University of Newcastle, Australia, New South Wales (ML); Division of Gastroenterology, Department of Medicine, St. Michael's Hospital, Toronto (YIK).
Correspondence to: Young-In Kim, MD, Medical Sciences Building, Rm. 7258, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 (e-mail: youngin.kim{at}utoronto.ca)
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ABSTRACT |
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INTRODUCTION |
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An accumulation of 5,10-methyleneTHF resulting from the MTHFR C677T polymorphism (6) may also have an effect on the response of cancer cells to two commonly used chemotherapeutic agents, 5-fluorouracil (5FU) and methotrexate (MTX), because the activity of both drugs is dependent on a competitive interaction with folate metabolism. 5,10-MethyleneTHF is the methyl donor for the nonreversible methylation, catalyzed by thymidylate synthase, of deoxyuridine-5'-monophosphate (dUMP) to deoxythymidine-5'-monophosphate (dTMP), a precursor for DNA synthesis (Fig. 1). 5,10-MethyleneTHF is also involved in de novo purine biosynthesis (Fig. 1).
Although, in general terms, 5FU is considered to be a folate antimetabolite, it has several potential cytotoxic mechanisms. Two metabolites of 5FU, 5-fluoro-2'-deoxyuridine-5'-triphosphate and 5-fluorouridine-5'-triphosphate, can be incorporated into DNA and RNA, respectively, resulting in DNA instability and interfering with RNA processing and function (9). 5FU can also form a ternary complex involving 5-fluoro-2'-deoxyuridine-5'-monophosphate (5FdUMP; the active metabolite of 5FU), thymidylate synthase, and 5,10-methyleneTHF. The formation of this complex thereby inhibits thymidylate synthase activity, which subsequently depletes intracellular thymidylate levels and ultimately suppresses DNA synthesis (Fig. 1) (9). Leucovorin (also known as folinic acid) or 5'-formylTHF, a precursor of 5,10-methyleneTHF, potentiates the cytotoxic effect of 5FU by stabilizing the inhibitory 5,10-methyleneTHFthymidylate synthase5FdUMP ternary complex (9). Therefore, the MTHFR C677T polymorphism, which increases intracellular concentrations of 5,10-methyleneTHF, may increase the cytotoxic effect of 5FU by increasing the formation and stability of the 5,10-methyleneTHFthymidylate synthase5FdUMP ternary complex.
By contrast, the cytotoxic effect of MTX may be compromised by an accumulation of intracellular 5,10-methyleneTHF resulting from the MTHFR C677T polymorphism. MTX inhibits dihydrofolate reductase, decreases intracellular 5,10-methyleneTHF levels for thymidylate biosynthesis, and directly inhibits purine biosynthesis. The accumulated dihydrofolate can inhibit both thymidylate synthase and enzymes involved in purine biosynthesis (Fig. 1) (10).
We hypothesized that the MTHFR C677T polymorphism would differentially modulate chemosensitivity to 5FU and MTX as a consequence of the increased 5,10-methyleneTHF pool. Because of the prevalence of the MTHFR C677T polymorphism and the fact that 5FU and MTX are widely used chemotherapeutic agents for the treatment of colon and breast cancers (9,10), two of the most common cancers globally, the MTHFR C677T polymorphism may be an important pharmacogenetic determinant of predicting response to 5FU and MTX. Identification of such a pharmacogenetic determinant would thereby enable physicians to provide rational and effective tailored chemotherapy to patients with colon and breast cancers. To test our hypothesis, we generated an in vitro model of the MTHFR C677T polymorphism in colon and breast adenocarcinoma cells and determined their chemosensitivity to 5FU and MTX.
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METHODS |
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Human colon adenocarcinoma HCT116 cells were purchased from the American Type Culture Collection (Manassas, VA), and human breast adenocarcinoma MDA-MB-435 cells were provided by Dr. M. Archer (University of Toronto, Toronto, Canada). Both cell lines were grown in RPMI-1640 medium (Invitrogen, Gaithersburg, MD) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin at 100 U/mL, and streptomycin at 100 mg/mL (complete medium). Cultures were maintained at 37 °C in 5% CO2.
MTHFR Genotyping and Construction and Transfection of MTHFR Expression Vectors
The analysis of the MTHFR C677T mutation in HCT116 and MDA-MB-435 cells was performed by real-time polymerase chain reaction (PCR) as described (11). We constructed three expression vectors for use in this study. The first vector expressed wild-type MTHFR, the second vector expressed a mutant 677T MTHFR, and the third vector expressed an antisense MTHFR that functioned as a transdominant negative control. The full-length human MTHFR (hMTHFR) cDNA (2.0 kilobases [kb]) was cloned from human colon adenocarcinoma Caco-2 cell total RNA by reverse transcription using the MTHFR-specific primer 5'-GGAGTGGCTCCAACGCAG-3' and followed by PCR amplification using the above primer as the antisense primer and 5'-AACCCAGCCATGGTGAAC-3' as the sense primer as described previously (12). The PCR product was first subcloned into the pBluescript SK(+) vector (Stratagene, Cambridge, U.K.). Four independent PCR clones were randomly selected and sequenced completely to avoid selecting clones with PCR-introduced sequence errors.
A thymine mutation at position 677 was introduced in the hMTHFR cDNA using PCR-based, site-directed mutagenesis (13). This method uses four primerstwo gene-specific (sense: 5'-AACCCAGCCATGGTGAAC-3'; antisense: 5'-CCTGGATGGGAAAGATCC-3') and two mutated (sense: 5'-AAGGT GTCTGCGGGAGTCGATTTCATCATCACGCAG-3'; antisense: 5'-GATGATGAAATCGACTCCCGCAGACACCTTCTCC-3'). Five nanograms of the hMTHFR cDNA was used as template, and 7.5 µM gene-specific primer and 0.75 µM mutated primer, respectively, were used. The wild-type and mutant 677T hMTHFR cDNAs were subcloned into the eukaryotic expression vector pIRESneo (Clontech, Palo Alto, CA) containing a CMV promoter and a neomycin resistance gene expression cassette. To generate the antisense vector that functioned as a positive control for impaired MTHFR expression, the full-length hMTHFR cDNA was subcloned into the pIRESneo vector in the antisense orientation. The correct integration, orientation, and sequences of the wild-type, mutant 677T, and antisense hMTHFR cDNAs were confirmed by predicted fragment sizes after multiple restriction enzyme digestions and DNA sequencing.
The pIRESneo vector containing the wild-type, mutant 677T, or antisense hMTHFR cDNA was stably transfected into HCT116 and MDA-MB-435 cells using Lipofectin (Invitrogen) according to the manufacturer's recommended protocol. In a separate transfection, HCT116 and MDA-MB-435 cells were stably transfected with empty pIRESneo (vector alone; endogenous hMTHFR). Transfected cells were incubated in the presence of neomycin (500 µg/mL; Invitrogen) to select for cells that expressed the various plasmids. After a population of cells was selected, individual clonal cell lines were isolated and expanded. Cells were maintained in complete medium supplemented with neomycin at 500 µg/mL. Several clones expressing the wild-type, 677T mutant, antisense hMTHFR cDNA, and empty vector from each cell line were selected at random for further analysis. Comparisons were made between cells expressing wild-type and 677T mutant hMTHFR and between cells expressing antisense and endogenous hMTHFR.
Western Blot Analysis
Total cellular lysates were obtained by incubating cells in RIPA solution containing protease inhibitors (phenylmethylsulfonyl fluoride at 0.1 mg/mL, aprotonin at 2 mg/mL) (Roche Diagnostics, Laval, Quebec, Canada). Supernatants were collected after centrifugation at 18 000g for 30 minutes at 4 °C. Protein concentrations were determined using a protein assay kit (Bio-Rad, Mississauga, Ontario, Canada). Fifty micrograms of total cellular protein from each cell line was separated on an 8% sodium dodecyl sulfatepolyacrylamide gel and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with phosphate-buffered saline (PBS) containing 5% skim milk for 2 hours at room temperature. To detect MTHFR protein expression, the membranes were incubated with a rabbit polyclonal antibody against amino acids 372395 of hMTHFR (Zymed, San Francisco, CA) at a dilution of 1 : 2000 in PBS containing 5% skim milk and 0.1% Tween 20 for 16 hours at 4 °C. To detect thymidylate synthase protein expression, the membranes were incubated with a sheep polyclonal antibody against human thymidylate synthase (Rockland Immunochemicals, Gilbertsville, PA) at a dilution of 1 : 3000 in PBS containing 5% skim milk and 0.1% Tween 20 for 16 hours at 4 °C. The MTHFR and thymidylate synthase proteins were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). To confirm that the proteins were loaded equally, the membranes were stripped and reprobed with a human anti--actin antibody (Sigma Aldrich Canada, Oakville, Ontario, Canada) at a dilution of 1 : 3000.
MTHFR Enzyme Assay
The 14C-labeled methyltetrahydrofolatemenadione oxidoreductase assay was used to measure specific MTHFR activity as described (14). Each assay used 100 µg of protein extract per cell line. The extracts were prepared as described (15). To determine MTHFR thermolability, the protein extracts were incubated at 46 °C for 5 minutes and residual MTHFR enzyme activity was measured as described (15).
Intracellular Folate Concentrations and Distributions
Cell pellets containing 20 x 106 cells were protected against labile folate oxidation by the addition of 100 µL of freshly made ascorbate (20 g/L). The cell suspension was then vortex mixed and stored in 100-µL aliquots at -80 °C until analyzed. Immediately before intracellular folate levels were measured, an aliquot of this cellascorbate preparation was thawed, and 50 µL of ascorbate (20 g/L) was added rapidly to the preparation and vortex mixed for 20 seconds. To this preparation, a total of 12.5 µL of 11M HClO4 was added, and the sample was mixed for 20 seconds. The preparation was then neutralized with the same volume of equimolar KOH, mixed, and spun at 13 000g for 2 minutes. The supernatant was collected, and 100 µL was injected into a liquid chromatograph for spectral confirmation of folate coenzyme identified using photodiode array detection based on the method of Lucock et al. (16). An isocratic high-performance liquid chromatography system with fluorescence detection (exc = 310 nm,
emm = 352 nm) (17) was used to quantify individual intracellular folate coenzyme species from 17.5 µL of supernatant. Intra-batch coefficient of variation (CV) values for the extraction and measurement of endogenous methylTHF and formylTHF polyglutamates (n = 8) were as follows: for methylTHF, CV = 4.70, 3.84, 2.94, 7.85, and 3.53 for polyglutamate chain lengths of 15 glutamate moieties, respectively; for formylTHF, CV = 18.82, 4.28, 4.13, 3.85, and 4.84 for polyglutamate chain lengths of 15 glutamate moieties, respectively.
Doubling-Time Calculation
Cells (8000 per well) were plated in 96-well plates and grown in RPMI-1640 medium with 10% fetal bovine serum for 72 hours. The cell population was determined using the sulforhodamine B (SRB) optical density (OD) measurement assay (18,19). The growth rate constant k was derived using an equation N/N0 = ekt, where N0 is the optical density of cells at time zero and N is the optical density of cells at 72 hours. The same equation was used to calculate the doubling time t by setting N/N0 = 2. All analyses were performed in triplicate, and three replicate experiments were performed.
In Vitro Chemosensitivity
In vitro chemosensitivity of cells stably transfected with the different MTHFR constructs was determined using a modified SRB protein assay as described (18,19). Briefly, 8000 cells per 100 µL medium per well were seeded in triplicate in 96-well flat-bottom plates. After 24 hours, an additional 100 µL of medium containing MTX (Schircks, Jona, Switzerland), 5FU alone, or 5FU (InvivoGen, San Diego, CA) in combination with leucovorin (Sigma Aldrich) was added, and the cells were cultured for an additional 72 hours. The concentration of MTX was varied, with concentrations ranging from 3.5 x 109M to 5 x 108M. The concentration of 5FU was varied, with concentrations ranging from 1.5 x 106M to 25 x 106M, whereas the concentration of leucovorin was held constant at 5 x 106M. Leucovorin was added to simulate the standard 5FU-based chemotherapy used in the treatment of colorectal and breast cancers (9). After 72 hours, cells were fixed with trichloroacetic acid and stained with SRB protein dye. The dye was solubilized, and the optical density of the solution measured at 595 nm. The results were expressed as a percentage of cell survival on the basis of the difference between the OD at the start and end of drug exposure, according to the formula (20):
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IC50 values (i.e., the drug concentration that corresponded to a reduction in cell survival by 50% compared with the survival of untreated control cells) were calculated from plots of drug concentration versus proportion of cells that survived.
In Vivo Chemosensitivity
Forty 6-week-old female BALB/c nu/nu mice (Charles River, Wilmington, MA) were randomly assigned to one of four groups (group 1, mice injected with xenografts expressing the wild-type MTHFR and treated with 0.9% NaCl; group 2, mice injected with xenografts expressing the mutant MTHFR and treated with 0.9% NaCl; group 3, mice injected with xenografts expressing the wild-type MTHFR and treated with 5FU; and group 4, mice injected with xenografts expressing the mutant MTHFR and treated with 5FU). Each group (n = 10) received a subcutaneous injection in the flank of HCT116 cells expressing wild-type or mutant 677T MTHFR (5 x 106 cells per mouse) in 0.2 mL serum-free RPMI-1640 medium. The cells were washed twice by centrifugation (1000g for 5 minutes) in serum-free RPMI-1640 medium before injection. When xenografts reached a volume of 80200 mm3 (approximately 3 weeks after injection), 5FU (40 mg/kg/day) dissolved in 0.2 mL of 0.9% NaCl was administered by intraperitoneal injection for 5 consecutive days a week for 2 weeks, as described (21). Leucovorin (4 mg/kg/day) was administered 1 hour before 5FU administration by intraperitoneal injection, as described (21). Control mice for each group received intraperitoneal injections of 0.9% NaCl. The tumors were measured with a caliper twice a week. The estimated tumor volume (V) was calculated by the following formula (22): V = W2 x L x 0.5, where W represents the largest tumor diameter in centimeters and L represents the next largest tumor diameter. The individual relative tumor volume (RTV) was calculated as follows: RTV = Vx/V1, where Vx is the volume in cubic millimeters at a given time and V1 is the volume at the start of treatment. Results are expressed as the mean daily percent change in tumor volume for each group of mice.
Thymidylate Synthase Catalytic Enzyme Activity
The catalytic activity of thymidylate synthase was determined by the 3H release that occurred during the conversion of [5-3H]-dUMP to dTMP, as described (23). Briefly, 10 µM [5-3H]-dUMP and 350 µM methyleneTHF (final concentration) were added to 150 µg of total cellular protein in a total volume of 50 µL of TrisHCl buffer (pH 7.4) for 1 hour at 37 °C. The reaction was ended by the addition of 50 µL of 35% trichloroacetic acid. After the addition of 250 µL of 10% activated charcoal in 0.2 M HCl, which bound the unreacted [5-3H]-dUMP, the mixture was centrifuged for 30 minutes, the supernatant was collected, and the amount of radioactivity in the supernatant was measured.
Statistical Analysis
Comparisons among cells expressing mutant and wild-type MTHFR were determined using Student's t test. For the in vitro chemosensitivity analyses, plots of percentage of survival versus dose demonstrated S-shaped curves, and therefore the logit transformation [logit(p) = ln(p/[1-p])] was used. Ordinary least-squares regression was used to model the effect of log(dose) of chemotherapy and cell type (wild-type versus mutant 677T MTHFR or antisense versus endogenous MTHFR) on the logit-transformed proportion of cells that survived at each dose. The interaction between cell type and log(dose) was included in the model to test the hypothesis that the cell types were differentially sensitive to chemotherapy. IC50 doses and their 95% confidence intervals (CIs) were calculated on the log-scale from the regression results, as described (24), and then back-transformed to the original scale for reporting. For the in vivo chemosensitivity analyses, because the tumor volumes were skewed, the data were log-transformed before analysis, producing a dependent outcome variable that was linear when plotted against time (measured in days). For each mouse, the rate of tumor growth was estimated using ordinary least-squares regression, with log(volume) as the dependent variable and day as the independent variable. The resulting slopes estimate the change in log(volume) per day and, when back-transformed, estimate the growth rate per day. The slope estimates were used as the dependent variable in a two-way analysis of variance. The two independent factors were tumor type (wild-type versus mutant 677T) and treatment (chemotherapy versus saline). An interaction effect was included in the analysis to test the hypothesis that the effect of chemotherapy differed for the two cell types. For all analyses, results were considered statistically significant if two-tailed P values were less than .05. Analyses were performed using SAS, version 8 (SAS Institute, Cary, NC).
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RESULTS |
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Parental HCT116 and MDA-MB-435 cell lines were both heterozygous (CT) for the MTHFR 677 genotype. Compared with untransfected parental cells, specific MTHFR enzyme activity and total intracellular folate concentrations from mock-transfected HCT116 and MDA-MB-435 cells were similar.
The wild-type and mutant 677T MTHFR proteins were abundantly expressed in HCT116 and MDA-MB-435 cells transfected with the wild-type and mutant 677T MTHFR cDNAs (Fig. 2, A). Specific MTHFR enzyme activity was approximately 35% lower (95% CI = 31% to 39%) in colon and breast cancer cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR protein (P<.001). In both HCT116 and MDA-MB-435 cells, the expression of the mutant 677T MTHFR was associated with statistically significantly higher thermolability than expression of the wild-type MTHFR, because there was statistically significantly lower residual MTHFR activity after heating cell lysates at 46 °C (Fig. 2, B and C; P<.001). As a positive control, HCT116 cells stably transfected with the vector containing the full-length hMTHFR cDNA in the antisense orientation were compared with HCT116 cells transfected with the vector without an insert. Both western analysis and the specific MTHFR enzyme assay confirmed that, compared with cells transfected with the vector alone, there was a statistically significant inhibition of MTHFR protein expression and activity in HCT116 cells transfected with the antisense vector (Fig. 2, A and D; P<.001).
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Both HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR grew faster than corresponding cells expressing the wild-type MTHFR. The doubling time of HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR was statistically significantly shorter than that for corresponding cells expressing the wild-type MTHFR (HCT116 cells, 35.4 ± 0.1 hours versus 38.2 ± 1.1 hours, P<.001; MDA-MB-435 cells, 45.0 ± 0.7 hours versus 52.4 ± 2.0 hours, P<.001). By contrast, growth of HCT116 cells expressing the antisense MTHFR was inhibited, and the doubling time statistically significantly increased compared with that for the corresponding cells expressing endogenous MTHFR (30.2 ± 0.8 hours versus 25.4 ± 0.2 hours; P<.001). The accelerated growth rate associated with the MTHFR 677T mutation is consistent with the known biochemical ramification of the MTHFR C677T polymorphism, which results in an accumulation of 5,10-methyleneTHF (6,7) and a consequent increase in thymidylate and purine biosynthesis. However, the inhibitory effect of antisense MTHFR on growth rate is not readily explained by the changes in intracellular folate composition associated with MTHFR inhibition.
Effect of the MTHFR 677T Mutation on Chemosensitivity of HCT116 and MDA-MB-435 Cells to 5FU In Vitro and In Vivo
We hypothesized that the MTHFR C677T polymorphism, which increases intracellular concentrations of 5,10-methyleneTHF, would enhance the cytotoxic effect of 5FU by increasing the formation and stability of the 5,10-methyleneTHFthymidylate synthaseFdUMP ternary complex. We first tested this hypothesis in an in vitro chemosensitivity assay. The chemosensitivity of HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR to 5FU plus leucovorin was statistically significantly increased compared with the corresponding cells expressing the wild-type MTHFR at each concentration of 5FU tested (P<.001 for both; Fig. 3, A and B). The IC50 value for 5FU was statistically significantly lower in HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR than in the corresponding cells expressing the wild-type MTHFR (4.2 µM [95% CI = 4.0 to 4.4 µM] versus 9.3 µM [95% CI = 8.8 to 9.8 µM] for HCT116 cells and 7.2 µM [95% CI = 6.6 to 7.8 µM] versus 31.6 µM [95% CI = 26.1 to 40.0 µM] for MDA-MB-435 cells; P<.001). In vitro chemosensitivity to 5FU plus leucovorin was statistically significantly increased in HCT116 cells expressing the antisense MTHFR compared with cells expressing endogenous MTHFR (P = .003; Fig. 3, C). The corresponding IC50 value was statistically significantly lower in HCT116 cells expressing the antisense MTHFR than in cells expressing endogenous MTHFR (1.7 µM [95% CI = 1.6 to 2.1 µM] versus 12.3 µM [95% CI = 10.7 to 14.3 µM]; P<.001).
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We next tested whether the MTHFR 677T mutation affected the in vivo chemosensitivity of HCT116 cells to 5FU plus leucovorin in nude mice. Leucovorin was added to simulate the standard 5FU-based chemotherapy used in the treatment of colorectal cancer and, thus, in vivo chemosensitivity to 5FU alone was not tested. When the growth rates of the mutant 677T MTHFR and wild-type xenografts in mice injected with saline were compared, it was evident that the mutant 677T MTHFR xenografts grew faster than the wild-type xenografts (average growth rate = 17.6%/day [95% CI = 12.7 to 22.7%/day] versus 9.3%/day [95% CI = 6.1 to 12.6%/day); P = .007; Fig. 4, A). The growth rate of the HCT116 xenografts expressing the mutant 677T MTHFR was inhibited more effectively by 5FU plus leucovorin (78% inhibition) than was the growth of those expressing the wild-type MTHFR (36% inhibition) (P = .008; Fig. 4, B). The in vivo chemosensitivity of these results support the in vitro observations that cancer cells expressing the mutant 677T MTHFR have faster growth rates than cells expressing wild-type MTHFR, but that cells expressing the mutant 677T MTHFR have increased sensitivity to 5FU plus leucovorin.
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We next hypothesized that the cytotoxic effect of MTX would be compromised by an accumulation of intracellular 5,10-methyleneTHF resulting from the MTHFR C677T polymorphism, because this would counteract the mode of MTX action, which depletes intracellular methyleneTHF for thymidylate and purine biosynthesis. We tested this hypothesis by comparing the in vitro chemosensitivity of HCT116 and MDA-MB-435 cells to MTX. In vitro chemosensitivity of MDA-MB-425 cells expressing the mutant 677T MTHFR to MTX was statistically significantly decreased compared with the chemosensitivity of cells expressing wild-type MTHFR (P = .011; Fig. 3, H). The IC50 value for MTX was statistically significantly higher in MDA-MB-435 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR (27.2 nM [95% CI = 22.5 to 34.5 nM] versus 8.6 nM [95% CI = 7.0 to 10.2 nM]; P<.001). By contrast, there was no statistically significant difference in chemosensitivity to MTX between HCT116 cells expressing the mutant 677T MTHFR and those expressing wild-type MTHFR (P = .98; Fig. 3, G). The IC50 value of MTX was not statistically significantly different between HCT116 cells expressing the mutant 677T MTHFR and those expressing wild-type MTHFR (3.7 nM [95% CI = 3.2 to 4.3 nM] versus 3.1 nM [95% CI = 2.6 to 4.0 nM]). However, in vitro chemosensitivity to MTX was statistically significantly decreased (P<.001; Fig. 3, I), and the IC50 value for MTX was statistically significantly increased (7.5 nM [95% CI = 7.0 to 8.1 nM] versus 6.2 nM [95% CI = 6.0 to 6.3 nM]; P<.001) for HCT116 cells expressing the antisense MTHFR compared with cells expressing the endogenous MTHFR. Consistent with the predictable changes in intracellular composition of folates, the MTHFR C677T polymorphism decreased chemosensitivity of breast cancer cells to MTX, whereas this effect was observed only with the antisense MTHFR, and not with the C677T mutation, in colon cancer cells.
Effect of the MTHFR 677T Mutation on Thymidylate Synthase Catalytic Activity in HCT116 and MDA-MB-435 Cells
We hypothesized that increased intracellular levels of 5,10-methyleneTHF resulting from the MTHFR 677T mutation would increase thymidylate synthase catalytic activity by providing abundant amounts of the methyl donor for the methylation, catalyzed by thymidylate synthase, of dUMP to dTMP (Fig. 1). Thymidylate synthase protein expression was similar between HCT116 and MDA-MB-435 cells expressing the wild-type MTHFR and cells expressing the mutant 677T MTHFR or between HCT116 cells expressing the antisense MTHFR and cells expressing endogenous MTHFR (Fig. 5, A). However, thymidylate synthase catalytic activity in HCT116 and MDA-MB-435 cells expressing the 677T mutant MTHFR was statistically significantly higher than in the corresponding cells expressing the wild-type MTHFR (P<.001; Fig. 5, B and C). Similarly, HCT116 cells expressing the antisense MTHFR had a statistically significantly higher level of thymidylate synthase catalytic activity than cells expressing endogenous MTHFR (P<.001; Fig. 5, D). Therefore, consistent with the changes in intracellular folate composition, the MTHFR C677T mutation and antisense inhibition increase thymidylate synthase catalytic activity but do not affect its protein expression.
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DISCUSSION |
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We hypothesized that increased intracellular concentrations of 5,10-methyleneTHF resulting from the MTHFR C677T polymorphism would increase the chemosensitivity of colon and breast cancer cells to 5FU by increasing the formation and stability of the 5,10-methyleneTHFthymidylate synthaseFdUMP ternary complex, with a consequent inhibition of thymidylate synthase and DNA synthesis. Furthermore, we hypothesized that cytotoxicity of MTX would be compromised by the MTHFR C677T polymorphism because of the increased intracellular concentrations of 5,10-methyleneTHF that ensure the flow of one-carbon units into thymidylate and purine biosynthesis. We developed an in vitro model of the MTHFR 677T mutation in colon and breast cancer cells with predictable functional consequences including decreased MTHFR activity, increased MTHFR thermolability, changes in the intracellular pool of folate derivatives (decreased methylTHF and increased formylTHF/methenylTHF), and increased thymidylate synthase activity. With this model, we showed that the MTHFR 677T mutation increased chemosensitivity of colon and breast cancer cells to 5FU and decreased chemosensitivity of breast cancer cells to MTX. For colon cancer cells, increased chemosensitivity to 5FU associated with the MTHFR 677T mutation was observed both in vitro and in vivo. There was a suggestion that the addition of leucovorin increased the chemosensitivity of colon and breast cancer cells expressing mutant 677T MTHFR to 5FU. The lack of effect of the MTHFR 677T mutation on chemosensitivity of colon cancer cells to MTX is not entirely surprising because colon cancer cells are generally not sensitive to MTX (26). Interestingly, however, antisense inhibition of MTHFR statistically significantly decreased chemosensitivity of colon cancer cells to MTX, raising the possibility that greater MTHFR inhibition than that achieved in our study may modulate chemosensitivity of colon cancer cells to MTX. Collectively, on the basis of the metabolic consequences of the changes in intracellular composition of folate derivatives, our study provides evidence that the MTHFR C677T polymorphism differentially modulates chemosensitivity of colon and breast cancer cells to chemotherapeutic agents, depending on their modes of action. Our data also suggest that antisense inhibition of MTHFR may be a potential target for increasing sensitivity of colon and breast cancer cells to 5FU-based chemotherapy.
Thymidylate synthase is a critical target for 5FU and MTX, and its expression level is an important prognostic factor for colorectal and breast cancers and a predictor of chemosensitivity of cancer cells to 5FU and MTX. Tumor thymidylate synthase expression appears to be inversely related to prognosis in patients with colorectal and breast cancers (25). Careful analyses of the literature and emerging new evidence, however, suggest that high thymidylate synthase expression is a poor prognostic factor for untreated patients with colorectal cancer but a good prognostic factor for patients treated with adjuvant 5FU-based chemotherapy (2730). By contrast, low thymidylate synthase expression seems to be a poor prognostic factor for patients treated with adjuvant 5FU-based chemotherapy (2730). A large series of in vitro preclinical data collectively suggest a positive association between thymidylate synthase activity and 5FU sensitivity in colorectal and breast cancer cell lines (3133). These in vivo (2730) and in vitro (3133) observations suggest that the most effective inhibition of thymidylate synthase will occur in cancer cells that are rapidly dividing and thus have high thymidylate synthase expression or activity. Our study indicates that the MTHFR 677T mutation and antisense MTHFR inhibition increase thymidylate synthase catalytic activity as a result of an increased supply of 5,10-methyleneTHF, the methyl donor for the methylation of dUMP to dTMP (Fig. 1). This mechanism is supported by the observation that thymidylate synthase protein expression was not statistically significantly different between colon and breast cancer cells expressing wild-type MTHFR and cells expressing mutant 677T MTHFR and between colon cancer cells expressing antisense MTHFR and cells expressing endogenous MTHFR. In cells treated with MTX, the MTHFR 677T mutation decreased chemosensitivity of breast cancer cells because the increased thymidylate synthase catalytic activity and intracellular 5,10-methyleneTHF concentrations enhanced thymidylate and purine biosynthesis, thereby counteracting the mode of MTX action. In cells treated with 5FU, the MTHFR 677T mutation increased chemosensitivity of colon and breast cancer cells because the increased 5,10-methyleneTHF concentrations and thymidylate synthase catalytic activity enhanced the formation and stability of the inhibitory 5,10-methyleneTFHthymidylate synthaseFdUMP ternary complex, thereby augmenting the mode of 5FU action.
Because the MTHFR C677T polymorphism occurs with an allelic frequency of about 35%, its role in cancer pharmacogenetics has important health implications. For this polymorphism to be used as a pharmacogenetic determinant in predicting response to and toxicities from chemotherapy, the observed effects of the MTHFR C677T polymorphism on sensitivity of colon and breast cancers to 5FU and MTX need to be confirmed in human studies. Recent human studies have suggested that the MTHFR TT genotype may increase toxicity to MTX alone or in combination with other chemotherapeutic agents in patients undergoing bone marrow transplantation (34), with leukemia (35), with ovarian cancer (36), and with breast cancer (37). Another study (38) involving 43 patients with metastatic colorectal cancer who received 5FU and other fluoropyrimidine-based chemotherapy has shown a statistically significant difference in the frequency of the T MTHFR allele among responders versus nonresponders (P = .035), with an odds ratio of 2.86 (95% CI = 1.06 to 7.73) for a response in individuals with a T allele. However, the differences in the proportion of objective responses among individuals with CC, CT, and TT genotypes did not reach statistical significance, likely because of the small sample size. Large clinical trials are therefore necessary to confirm the effect of the MTHFR C677T polymorphism on treatment response and survival in cancer patients receiving chemotherapy.
Although colon cancer cells expressing mutant 677T MTHFR and cells expressing antisense MTHFR had similar biochemical consequences, only cells expressing mutant 677T MTHFR had an accelerated growth rate. This discrepancy in growth rate between cells expressing 677T MTHFR and antisense MTHFR is unclear. One possible explanation is related to total intracellular folate concentrations. Colon cancer cells expressing antisense and endogenous MTHFR had statistically significantly lower total intracellular folate concentrations than cells expressing wild-type and mutant 677T MTHFR. Relative to changes in the appropriate control cell populations, folate depletion in colon cancer cells expressing antisense MTHFR was greater than that in cells expressing mutant 677T MTHFR. Therefore, it is possible that the relative depletion of total intracellular folates might explain the observed growth inhibition associated with the antisense MTHFR, overriding the effects of an increased relative proportion of 5,10-methyleneTHF and increased thymidylate synthase activity.
The effect of the MTHFR C677T polymorphism on total tissue folate concentrations and intracellular folate composition, particularly in the target organs such as the colon and breast, has not been reported. Generally, the MTHFR C677T polymorphism has been associated with lower plasma folate concentrations than the wild-type genotype (25). However, data are equivocal for the effect of the MTHFR C677T polymorphism on folate concentrations in red blood cells (3,6,7,3941), although the MTHFR C677T polymorphism does appear to decrease methylTHF and increase formylTHF in red blood cells (6,42). In a recent study (43), no statistically significant differences in total folate content were observed in liver and brain tissues among wild-type (Mthfr+/+), heterozygous (Mthfr+/), and knockout (Mthfr/) mice, although the proportion of methylTHF in liver and brain tissues was statistically significantly lower in knockout mice than in wild-type mice. Our data show that, compared with the wild-type MTHFR, the MTHFR C677T polymorphism is associated with a decreased relative proportion of methylTHF and an increased relative proportion of formylTHF/methenylTHF (and thus a higher proportion of methyleneTHF) in human colon and breast cancer cells. However, our data demonstrate that, compared with the wild-type MTHFR, the MTHFR C677T polymorphism is associated with lower total folate concentrations in breast, but not colon, cancer cells. Collectively, our data and data from another study (43) suggest that the effect of the MTHFR C677T polymorphism on total tissue folate concentrations may be highly variable and tissue-specific.
One potential limitation of our study is that the functional effects of the MTHFR 677T mutation were determined in cells expressing endogenous MTHFR (i.e., HCT116 and MDA-MB-435 cells were heterozygous [CT] for MTHFR). However, comparisons were made between cells expressing the mutant 677T and wild-type MTHFRboth of which were statistically significantly overexpressedlikely overshadowing any effect of endogenous MTHFR. Furthermore, the functional effects of the MTHFR 677T mutation observed in our in vitro system were confirmed in an in vitro system of antisense MTHFR inhibition and in an in vivo model.
In conclusion, we provide evidence that the MTHFR C677T polymorphism may be an important pharmacogenetic determinant of 5FU- and MTX-based cancer chemotherapy. Our data suggest that the MTHFR C677T polymorphism may be a useful pharmacogenetic determinant for providing rational and effective tailored cancer chemotherapy. Furthermore, our data suggest that antisense inhibition of MTHFR may be a potential target for increasing chemosensitivity of colon and breast cancer cells to 5FU-based chemotherapy. The pharmacogenetics of the MTHFR C677T polymorphism may also be applied to other disease processes in which MTX, 5FU, and newer analogs are used for treatment (e.g., MTX in rheumatoid arthritis and inflammatory bowel disease).
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Manuscript received May 29, 2003; revised November 13, 2003; accepted December 4, 2003.
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