Methotrexate enhances 5-aminolevulinic acid-mediated photodynamic therapy-induced killing of human SCC4 cells by upregulation of coproporphyrinogen oxidase.
OBJECTIVE
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/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.


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.


CONCLUSIONS
MTX enhances ALA-PDT-induced SCC4 cell killing through upregulation of CPOX expression and subsequent increase in intracellular protoporphyrin IX production in SCC4 cells.
Graduate Program, Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan, Taiwan.
Deng-Fu
DF
Yang
Department of Life Science, Tzu Chi University, Hualien, Taiwan.
Jeng-Woei
JW
Lee
Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan.
Hsin-Ming
HM
Chen
Cancer Center, University of Colorado Denver Cancer Center, CO, USA.
Zheng
Z
Huang
Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan, Taiwan; Center for Nanotechnology, Chung Yuan Christian University, Taoyuan, Taiwan; Center of Biomedical Technology, Chung Yuan Christian University, Taoyuan, Taiwan. Electronic address: ivyhsu@cycu.edu.tw.
Yih-Chih
YC
Hsu
D000622:Aminolevulinic Acid / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy*; D003304:Coproporphyrinogen Oxidase / Q000378:metabolism*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D009062:Mouth Neoplasms / Q000188:drug therapy*; D010778:Photochemotherapy / Q000379:methods*; D011230:Precancerous Conditions / Q000188:drug therapy*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000622:Aminolevulinic Acid; D003304:Coproporphyrinogen Oxidase; D008727:Methotrexate
5-aminolevulinic acid; coproporphyrinogen oxidase; ferrochelatase; human squamous cell carcinoma 4 cell; methotrexate; photodynamic therapy