An in Vitro Study of the Effect of 5-ALA-mediated Photodynamic Therapy on Oral Squamous Cell Carcinoma

Ying Ma; Shujuan Qu; Liangpeng Xu; Hongbo Lu; Baoguo Li

BMC Oral Health. 2020;20(258)

Results

Comparison of Average Fluorescence Intensity of Intracellular Protoporphyrin IX in SCC25 Cells of Different Groups With Different Concentrations of 5-ALA for Different Lenths of Time

Table 1 shows that the level of protoporphyrin IX in SCC25 cells increased with increases in 5-ALA concentration and prolongation of time. However, after 12 h, the protoporphyrin IX level in SCC25 cells were gradually stabilized, and similar effect was obtained after using 100 mg/L or more 5-ALA, indicating that the level of protoporphyrin IX in SCC25 cells was determined by 5-ALA concentration and time.

Comparison of the Survival rate of SCC25 Cells Treated With 5-ALA and Laser for Different Time Between Groups

Laser irradiation alone, or 5, 10, 25, 50 or100 mg/L 5-ALA exerted no effect on cell viability, and the cell survival rate was 100%. 5-ALA combined with laser irradiation inhibited the growth of SCC25 cells, which was highly associated with drug concentration and time. Laser irradiation after co-incubation of 5-ALA with SCC25 cells for 12 h showed significant cytotoxic effect. When the concentration of 5-ALA was 50 mg/L, the cell survival rate was (8.97 ± 2.97) mg/L (Table 2).

Effects of 5-ALA Combined With Laser Irradiation on Apoptosis and Necrosis of SCC25 Cells in Each Group

Compared with the blank control group, laser irradiation alone and 5-ALA alone showed no effect on the apoptosis of SCC25 cells, and no significant difference was found in the early apoptosis rate, late apoptosis rate and necrosis rate of SCC25 cells (P > 0.05). Different concentrations of 5-ALA combined with laser irradiation showed a significant effect on apoptosis. A higher apoptosis rate was found with higher drug concentrations, which was significantly higher than that in the control group (P < 0.05) (Table 3).

Effect of 5-ALA Combined With Laser Irradiation on SCC25 Cells

Significant difference was observed in the effect of 5-ALA combined with laser irradiation compared with the blank control group (P < 0.05) (Table 4).

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Table 1. 5-ALA triggered OSCC cells to produce protoporphyrin IX (x̅ ± s, × 10 ³)
Group (5-ALA)	2 h	4 h	6 h	12 h	24 h
Blank	0.007 ± 0.001	0.005 ± 0.001	0.008 ± 0.002	0.018 ± 0.003	0.026 ± 0.003
5 mg/L	0.238 ± 0.006*	0.361 ± 0.012*	0.351 ± 0.017*	0.693 ± 0.037*^#	2.673 ± 0.048^#
10 mg/L	0.612 ± 0.061*	0.816 ± 0.028*	0.735 ± 0.123*^#	1.487 ± 0.067*^#	5.432 ± 0.079*^#
25 mg/L	2.557 ± 0.161*	3.212 ± 0.037*	5.721 ± 0.483*^#	11.987 ± 0.654*^#	16.987 ± 2.391*^#
50 mg/L	4.962 ± 0.127*	7.791 ± 0.263*	11.723 ± 1.273*^#	21.076 ± 0.763*^#	27.473 ± 3.473*^#
100 mg/L	7.643 ± 1.673*	11.376 ± 2.065*^#	14.723 ± 1.238*^#	26.753 ± 2.632*^#	32.763 ± 3.973*^#
150 mg/L	8.124 ± 1.721*	12.451 ± 2.376*^#	16.302 ± 1.098*^#	29.876 ± 2.398*^#	33.902 ± 4.872*^#

The sample size at each time point was 3 in each group; compared with the blank group at the same time point, *P < 0.05; compared with the same group after 2 h, #P < 0.05

Table 2. Comparison of the survival rate of SCC25 cells treated with 5-ALA and laser for different time between group (x̅ ± s,%)
Group (5-ALA)	2 h	4 h	6 h	12 h
Blank	102.01 ± 7.03	98.16 ± 6.93	97.37 ± 6.51	95.97 ± 6.19
5 mg/L	98.08 ± 7.17	94.09 ± 6.09	87.92 ± 7.89	81.92 ± 4.99
10 mg/L	95.83 ± 6.83	93.81 ± 6.19	89.36 ± 4.87	75.82 ± 3.91*^#
25 mg/L	82.13 ± 3.01*	76.89 ± 3.98*	66.87 ± 8.79*^#	37.92 ± 8.73*^#
50 mg/L	62.01 ± 8.02*	57.93 ± 8.35*	41.28 ± 7.32*^#	8.97 ± 2.97*^#
100 mg/L	78.82 ± 6.53*	53.73 ± 4.83*	29.76 ± 5.83*^#	7.19 ± 1.26*^#

The sample size at each time point was 6 in each group; compared with the blank group at the same time point, *P < 0.05; compared with the same group after 4 h, #P < 0.05

Table 3. Effect of 5-ALA combined with laser irradiation on apoptosis rate and necrosis rate of SCC25 cells in each group (x̅ ± s, %)
Group (5-ALA)	Early apoptosis rate	Late apoptosis rate	Necrosis rate
Blank	0.49 ± 0.07	0.33 ± 0.11	0.39 ± 0.19
100 mg/L	0.51 ± 0.13	0.44 ± 0.17	0.55 ± 0.26
Laser	0.63 ± 0.17	0.35 ± 0.08	0.48 ± 0.23
Laser + 5 mg/L	1.41 ± 0.36*	3.09 ± 0.31^#	1.38 ± 0.34*
Laser + 10 mg/L	1.18 ± 0.35*	5.29 ± 0.49^#	2.73 ± 0.28^#
Laser + 25 mg/L	7.83 ± 0.98^#	17.87 ± 3.01^#	7.41 ± 1.23^#
Laser + 50 mg/L	5.47 ± 0.76^#	24.54 ± 4.02^#	13.89 ± 2.01^#
Laser + 100 mg/L	24.87 ± 4.01^#	44.28 ± 6.73^#	14.83 ± 3.03^#

The sample size at each time point was 3 in each group; compared with the blank group at the same time point, *P < 0.05; compared with the same group after 4 h, #P < 0.05

Table 4. Effect of 5-ALA combined with laser irradiation on SCC25 cell cycle (x̅ ± s)
Group/Stage (%)	I	II	III	IV
Blank	55.832 ± 0.213	3.2876 ± 0.098	5.987 ± 0.211	39.765 ± 0.238
100 mg/L 5-ALA+Laser	78.932 ± 0.238^#	2.473 ± 0.029^#	4.087 ± 0.231^#	17.231 ± 0.029^#