Effects of folylpolyglutamate synthetase modulation on chemosensitivity of colon cancer cells to 5-fluorouracil and methotrexate

Abstract

Background: Folylpoly-gamma-glutamate synthetase (FPGS) converts intracellular folates and antifolates (for example, methotrexate (MTX)) to polyglutamates. Polyglutamylated folates and antifolates are retained in cells longer and are better substrates than their monoglutamate counterparts for enzymes involved in one carbon transfer. Polyglutamylation of intracellular 5,10-methylenetetrahydrofolate may also enhance the cytotoxicity of 5-fluorouracil (5-FU) by allowing more efficient formation and stabilisation of the inhibitory ternary complex involving thymidylate synthase and a 5-FU metabolite.

Aim: We investigated the effects of FPGS modulation on the chemosensitivity of colon cancer cells to 5-FU and MTX.

Methods: Human HCT116 colon cancer cells were stably transfected with the sense or antisense FPGS cDNA or blank (control). FPGS protein expression and enzyme activity, growth rate, intracellular folate content and composition, and in vitro chemosensitivity to 5-FU and MTX were determined.

Results: Compared with cells expressing endogenous FPGS, those overexpressing FPGS had significantly faster growth rates and higher concentrations of total folate and long chain folate polyglutamates while antisense FPGS inhibition produced opposite results. FPGS overexpression significantly enhanced, whereas FPGS inhibition decreased, chemosensitivity to 5-FU. No significant difference in chemosensitivity to MTX was observed.

Conclusions: These data provide functional evidence that FPGS overexpression and inhibition modulate chemosensitivity of colon cancer cells to 5-FU by altering intracellular folate polyglutamylation, providing proof of principle. Thus FPGS status may be an important predictor of chemosensitivity of colon cancer cells to 5-FU based chemotherapy, and FPGS gene transfer may increase the sensitivity of colon cancer cells to 5-FU-based chemotherapy.

Figure 1

HCT116 colon cancer cells transfected with the sense (FPGS-S) and antisense (FPGS-AS) folylpolyglutamate synthetase (FPGS) cDNA had significantly higher and lower steady state levels of FPGS protein (A) and FPGS activity (B), respectively, compared with cells transfected with vector alone (VA; endogenous FPGS) (p<0.001).

Figure 2

Intracellular folate concentrations of HCT116 cells expressing the sense (FPGS-S), endogenous (vector alone (VA)), and antisense (FPGS-AS) folylpolyglutamate synthetase (FPGS). Conjugase treatment allows measurement of total folate content, including short and long chain polyglutamates, whereas non-conjugase treatment determines the content of short chain polyglutamates. Intracellular concentrations of total folate were significantly higher in cells expressing the sense FPGS and lower in cells expressing the antisense FPGS, respectively, than in those expressing endogenous FPGS (p<0.0001). Differences between mean folate concentration for conjugase treated and untreated samples (which allows determination of long chain polyglutamates) were significantly higher in cells expressing the sense FPGS and lower in cells expressing the antisense FPGS, respectively, compared with those expressing endogenous FPGS (p<0.001).

Figure 3

HCT116 cells expressing the sense folylpolyglutamate synthetase (FPGS-S) had significantly higher, whereas those expressing the antisense FPGS (FPGS-AS) had significantly lower, γ-glutamyl hydrolase (GGH) mRNA expression compared with those expressing endogenous FPGS (vector alone (VA)) (p<0.05), as determined by real time quantitative reverse transcription-polymerase chain reaction. GGH is a lysosomal peptidase that removes the terminal glutamates, thereby counterbalancing the action of FPGS. Human 5-aminolevulinate delta synthase 1 was used as the endogenous reference gene (primers: forward, 5′-TGC CCA TTC TTA TCC CGA GT-3′; reverse, 5′-GGT TTC TTT GAT CTG TTG GTA GTG-3′). Relative quantification was performed using LC Relative Quantification Software version 1.0 (Roche Diagnostics).

Figure 4

(A) In vitro chemosensitivity to 5-fluorouracil (5-FU) plus leucovorin (LV) was significantly different among HCT116 colon cancer cells expressing the sense (FPGS-S), endogenous (vector alone (VA)), and antisense (FPGS-AS) folylpolyglutamate synthetase (FPGS) (p<0.001). Chemosensitivity of cells expressing the sense FPGS to 5-FU+LV was significantly enhanced compared with those expressing endogenous or the antisense FPGS (p<0.0001). In contrast, chemosensitivity of cells expressing the antisense FPGS was significantly decreased compared with those expressing endogenous or the sense FPGS (p = 0.0122 and p<0.0001, respectively). (B) In vitro chemosenstivity to methotrexate (MTX) was not significantly different among HCT116 cells expressing the sense, endogenous, and antisense FPGS (p = 0.548).

Figure 5

Effect of folylpolyglutamate synthetase (FPGS) modulation on p53 and p21. p53 is integrally involved in cell cycle control, DNA repair, and apoptosis while p21 is a member of the cyclin dependent kinase inhibitor family, which inhibits G₁ and S phase progression. p53 promotes cell cycle arrest in late G₁ through upregulation of p21 in response to cell injury, thereby allowing time for repair, whereas in the event that DNA damage is more severe and non-reparable, p53 performs its alternative role of moving the cell into apoptosis. FPGS overexpression (FPGS-S) downregulated, whereas FPGS inhibition (FPGS-AS) upregulated, p53 and p21 protein expression compared with endogenous FPGS (vector alone (VA)) in HCT116 cells (densitometry of bands: p53:β-actin ratio 1.9 (FPGS-S) v 3.1 (VA) v 4.1 (FPGS-AS); p21:β-actin ratio 1.7 (FPGS-S) v 2.5 (VA) v 3.2 (FPGS-AS)).