To measure the SCC4 cell killing abilities by MTX-pretreated ALA-PDT (MTX-ALA-PDT), the SCC4 cells were pretreated with 0聽mg/L, 0.001聽mg/L, 0.01聽mg/L, 0.1聽mg/L, or 1聽mg/L of MTX for 72 hours, then incubated with 0聽mM, 0.0625聽mM, 0.125聽mM, 0.187聽mM, 0.25聽mM, or 0.375聽mM ALA for 4 hours, and subsequently illuminated with a 640-nm light-emitting diode array at a light dose of 10聽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.
Pretreatment of SCC4 cells by 0.001聽mg/L MTX for 72 hours resulted in a significant augmentation in MTX-ALA-PDT-induced killing of SCC4 cells (p聽<聽0.05). The SCC4 cells treated with 0.001聽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聽<聽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.