Abstract
Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is effective for treatment of oral precancerous and cancerous lesions. This invitro study tried to examine whether the SCC4 cell killing by ALA-PDT was enhanced by pretreatment of methotrexate (MTX). To measure the SCC4 cell killing abilities by MTX-pretreated ALA-PDT (MTX-ALA-PDT), the SCC4 cells were pretreated with 0mg/L, 0.001mg/L, 0.01mg/L, 0.1mg/L, or 1mg/L of MTX for 72 hours, then incubated with 0mM, 0.0625mM, 0.125mM, 0.187mM, 0.25mM, or 0.375mM ALA for 4 hours, and subsequently illuminated with a 640-nm light-emitting diode array at a light dose of 10J/cm(2). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted at 24 hours to quantify SCC4 cell survival rates after MTX-ALA-PDT treatment. Western blot analyses were used to examine the MTX-mediated enhancement in the expressions of the heme production-related enzymes, coproporphyrinogen oxidase (CPOX), protoporphyrinogen oxidase (PPOX), and ferrochelatase, in the MTX-preconditioned SCC4 cells. Pretreatment of SCC4 cells by 0.001mg/L MTX for 72 hours resulted in a significant augmentation in MTX-ALA-PDT-induced killing of SCC4 cells (p<0.05). The SCC4 cells treated with 0.001mg/L MTX for 72 hours showed a significant and 1.65-fold increase in CPOX expression compared with the control SCC4 cells without MTX treatment (p<0.05). However, no significant changes in the expressions of PPOX and ferrochelatase were observed in the SCC4 cells pretreated with different concentrations of MTX. MTX enhances ALA-PDT-induced SCC4 cell killing through upregulation of CPOX expression and subsequent increase in intracellular protoporphyrin IX production in SCC4 cells.
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