| Summary: | Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is effective for treatment of oral precancerous and cancerous lesions. This in vitro study tried to examine whether the SCC4 cell killing by ALA-PDT was enhanced by pretreatment of methotrexate (MTX).
Methods: To measure the SCC4 cell killing abilities by MTX-pretreated ALA-PDT (MTX–ALA-PDT), the SCC4 cells were pretreated with 0 mg/L, 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, or 1 mg/L of MTX for 72 hours, then incubated with 0 mM, 0.0625 mM, 0.125 mM, 0.187 mM, 0.25 mM, or 0.375 mM ALA for 4 hours, and subsequently illuminated with a 640-nm light-emitting diode array at a light dose of 10 J/cm2. 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.
Results: Pretreatment of SCC4 cells by 0.001 mg/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 0.001 mg/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.
Conclusion: 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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