Oxidative stress and inhibition of nitric oxide generation underlie methotrexate-induced senescence in human colon cancer cells

Abstract

The response of human colon cancer C85 cells to methotrexate takes the form of reversible growth arrest of the type of stress-induced senescence. In the present study it is shown that during C85 cell progression into methotrexate-induced senescence, dihydrofolate reductase, the primary intracellular target for the drug, is stabilized at the protein level and its enzymatic activity, assayed in crude cellular extracts, decreases by 2-fold. Dihydrofolate reductase inhibition results in an increase in dihydrobiopterin level and an ultimate decrease in the tetrahydrobiopterin: dihydrobiopterin ratio in senescent cells. Endothelial nitric oxide synthase expression declines. Despite concomitant upregulation of inducible nitric oxide synthase expression, no nitric oxide generation in senescent cells is detected. Progressing oxidative stress accompanies establishment of the state of senescence. DNA damage, in the form of double strand-breaks, occurs at the highest level at the senescence initiation phase and decreases as cells progress into the senescence maintenance phase.

Keywords: Colorectal cancer; Dihydrofolate reductase; Methotrexate; Nitric oxide signaling; Therapy-induced senescence.