Low-dose methotrexate enhances cycling of highly anaplastic cancer cells

We previously showed that cellular RedOx state governs the G₁-S transition of AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem cell stage. This transition is impaired when the mithocondrial electron transport system is blocked by specific inhibitors (antimycin A) or the respiratory chain is saturated by adding to the cells high concentrations of pyruvate. The antimycin A or pyruvate block is removed by the addition of adequate concentrations of folate (F). This suggests that the G₁-S transition of AH130 cells depends on a respiration-linked step of DNA synthesis related to folate metabolism. In the study reported here, we characterized the effects of methotrexate (MTX), an inhibitor of dihydofolate-reductase, on the G₁-S transition of hepatoma cells, in the absence or the presence of exogenously added F, dihydrofolate (FH₂) or tetrahydrofolate (FH₄). MTX, at 1 μM or higher concentrations, inhibited G₁-S transition. This inhibition was completely removed by exogenous folates. Surprisingly, 10 nM MTX stimulated G₁-S transition. The addition of F, but not FH₂ or FH₄, significantly increased this effect. Furthermore, 10 nM MTX removed the block of the G₁-S transition operated by antimycin A or pyruvate, an effect which was enhanced in the presence of F. Finally, the stimulatory effect of 10 nM MTX was inhibited in the presence of serine. Our findings indicated that, under certain conditions, MTX may stimulate, rather than inhibiting, the cycling of cancer cells exhibiting a stem cell-like phenotype, such as AH130 cells. This may impact the therapeutic use of MTX and of folates as supportive care.