[In vitro and in vivo effects of 5-aminolevulinic acid-mediated photodynamic therapy against oral squamous cell carcinoma]

Abstract

Objective: To investigate the in vitro and in vivo effects of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy against oral squamous cell carcinoma (OSCC) and preliminarily explore the possible mechanisms. Methods: SCC25 cells were divided into the control group (5-ALA of 0 mg/L) and the experimental group (5-ALA of 10, 25, 50, 100 and 150 mg/L). The production of protoporphyrin Ⅸ (PpⅨ) induced by 5-ALA in SCC25 cells was detected using the flow cytometry. SCC25 cells were divided into the control group (5-ALA of 0 mg/L), lazer alone group, 5-ALA alone group (5-ALA of 100 mg/L) and the 5-ALA combined with laser irradiation group (5-ALA of 5, 10, 25, 50 and 100 mg/L), the cytotoxicity of 5-ALA combined with laser irradiation (wave length 635 nm, power density 87 mW/cm(2) and laser dose 10.4 J/cm(2)) was evaluated in SCC25 cells using the methyl thiazolyltetrazolium assay (incubation times of 4, 8 and 12 h in each group) and the induction effect of combination treatment on the cell apoptosis was assessed by the flow cytometry (incubation time of 12 h in each group). The intracellular production of reactive oxygen species (ROS) triggered by 5-ALA combined with laser irradiation was determined using a fluorescence probe method (incubation time of 12 h in each group). A mouse OSCC xenograft model bearing SCC25 tumor was built, and the mice were divided into control group (saline), 5-ALA group (5-ALA of 50 mg/kg) and 5-ALA combined with laser irradiation group (5-ALA of 10, 25 and 50 mg/kg). Antitumor effect of 5-ALA combined with laser irradiation (wave length 635 nm, power density 158 mW/cm(2) and laser dose 94.8 J/cm(2)) was further measured. Results: 5-ALA induced the production of PpⅨ in SCC25 cells in a drug concentration (0-150 mg/L)-and incubation time (0-24 h)-dependent manner. When the 5-ALA concentration was 100 mg/L, the intracellular PpⅨ production was in a relatively stable state. Cell viability and apoptosis rate of 5, 10, 25, 50, 100 mg/L 5-ALA combined with laser irradiation are, respectively, (82.3±5.2)%, (3.13±0.38)%; (74.6±9.3)%, (5.38±0.55)%; (38.3±9.7)%, (17.97±2.72)%; (9.2±3.8)%, (24.47±3.37)%; (7.2±0.8)%, (43.01±5.96)%, which indicated that 5-ALA combined with laser irradiation notably inhibited the growth of SCC25 cells and also induced significant cell apoptosis compared with the control group [(96.3±6.0)%, (0.35±0.13)%, P<0.05]. After combination treatment (5-ALA of 5, 10, 25, 50 and 100 mg/L combined with laser irradiation, the mean fluorescence intensity of dichlorofluorescein is (1.46±0.12)×10(4), (2.16±0.30)×10(4), (3.57±0.34)×10(4), (81.70±13.05)×10(4), (113.00±7.35)×10(4), respectively, a large amount of ROS was produced in SCC25 cells compared with the control group [(0.96±0.15) ×10(4), P<0.05], which was in positive correlation with the intracellular PpⅨ content. 5-ALA (concentration of 10, 25 and 50 mg/kg) combined with laser irradiation greatly suppressed the tumor growth in SCC25 tumor-bearing mice compared to the control group (P<0.05). Conclusions: 5-ALA-mediated photodynamic therapy can trigger the generation of intracellular ROS that has significant cytotoxicity and apoptosis induction effect, and thus inhibit the tumor growth both in vitro and in vivo.

Keywords: 5-Aminolevulinic acid; Apoptosis; Carcinoma, squamous cell; Photochemotherapy.