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


OBJECTIVE
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.
Sohn
K-J
KJ
Medical Sciences Building, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
Smirnakis
F
F
Moskovitz
D N
DN
Novakovic
P
P
Yates
Z
Z
Lucock
M
M
Croxford
R
R
Kim
Y-I
YI
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology*; D004305:Dose-Response Relationship, Drug; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D005472:Fluorouracil; D008727:Methotrexate