Cobalamin potentiates vinblastine cytotoxicity through downregulation of mdr-1 gene expression in HepG2 cells.
BACKGROUND
P-glycoprotein (Pgp), produced by multidrug resistance-1 gene (mdr-1), is a main mechanism developed by cancer cells to guard against anti-cancer drugs. Alterations of DNA methylation of the mdr-1 gene promoter are known to be linked to mdr-1 gene expression and are probably related to intracellular S-adenosyl-methionine. We here used HepG2 cells to determine the role of the methionine cycle (through the use of the Methionine-Synthase (MS) cofactor, cobalamin) on mdr-1 gene expression.


METHODS
Semiquantitative RT-PCR of mdr-1 gene, cellular retention of rhodamine-123, and vinblastine cytotoxicity were carried out on cells cultivated with and without cobalamin. Methylation status of the mdr-1 gene promoter was determined by methylation-specific PCR.


RESULTS
Addition of cobalamin to the cells led to an increase in MS activity, to a significant decrease in mdr-1 gene expression which is correlated to an increase in retention of the Pgp substrate Rhodamine 123. Furthermore, cobalamin potentiated cell sensitivity to vinblastine to the same range as that of the Pgp blocker verapamil and prevented methotrexate-induced up-regulation of mdr-1 gene expression. However, no modification in methylation of the mdr-1 gene promoter was observed.


CONCLUSIONS
Cobalamin downregulates mdr-1 gene expression, as well as Pgp expression and function, and significantly increases cytotoxicity of vinblastine. The identification of this novel way of diminishing cellular resistance to the chemotherapeutic agent vinblastine holds promises of leading to better treatments for cancer patients.
Marguerite
Véronique
V
Laboratoire de Pathologie Cellulaire et Moleculaire en Nutrition, Faculté de Médecine, University Henry Poincaré, Vandoeuvre-les-Nancy, Cedex, France.
Beri-Dexheimer
Myléne
M
Ortiou
Sandrine
S
Guéant
Jean-Louis
JL
Merten
Marc
M
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004357:Drug Synergism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D020112:Rhodamine 123 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014747:Vinblastine / Q000494:pharmacology*; D014805:Vitamin B 12 / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020112:Rhodamine 123; D014747:Vinblastine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12; D008727:Methotrexate