Figure 3.

Effect of MTX upon ALA-mediated PpIX accumulation. (A) Pretreatment of LNCaP cells with MTX enhances ALA-induced PpIX accumulation. Cells were cultured in the presence of MTX (1 mg l⁻¹; grey bar) for 72 h, then ALA (0.3 mM) was added for 4 h, and cells harvested for measurements of intracellular PpIX. For a positive control, some dishes were incubated with an androgen, R1881 (0.1 μM; black bar) for 72 h. Protoporphyrin IX concentrations, calculated from a standard curve, were expressed on a per cell basis. Bars are the mean of eight experiments±s.e.m. (^*), two-sample t-test, P<0.001. (B) Methotrexate enhances PpIX accumulation in LNCaP cells over a range of ALA concentrations. Cells were pretreated with MTX (open circles) or medium alone (solid circles) for 72 h, then incubated for 4 h in different ALA concentrations before harvest. This experiment shows mean±range of duplicate determinations at each point. Two additional experiments gave qualitatively similar results (not shown). (Inset) Enlargement of the graph at 0.3 mM ALA; note that MTX enhancement of PpIX occurred even at the lowest ALA dose. (Asterisks) Two-sample t-test, comparing MTX to control at the same dose of ALA; (^*) P<0.05; (^**) P<0.001. (C) Effect of MTX upon accumulation of PpIX derived from different ALA esters. Cells were incubated for 72 h in the absence or presence of MTX, and then for 4 h in the presence of either ALA (0.3 mM), methyl-ALA (0.5 mM), or hexyl-ALA (1 μM) before harvest and PpIX measurement. Bars are the mean±s.e.m. for three experiments, each performed in duplicate. Numbers above each bracket indicate relative increase (-fold) in PpIX owing to MTX. Note the similar -fold increase with all three analogues.