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)

Background

Oral squamous cell carcinoma (OSCC) is the one of the most common malignant tumors with a high prevalence rate and a poor prognosis, which poses a serious threat to the life and health of patients. At present, surgery, adjuvant radiotherapy and chemotherapy are regarded as the main approaches to treat OSCC.^[1,2] As the conventional surgical method has some limitations, it cannot be widely used. On the other hand, radiotherapy and chemotherapy have toxic and side effects to some extent, and cancer cells could be induced to produce drug resistance during the operation, which reduces the efficacy of chemotherapy. Therefore, there has been a high demand for clinical research in recent years to adopt safe, effective and less limited treatment methods for OSCC. Photodynamic therapy is a therapeutic approach using photosensitive drugs combined with light irradiation to selectively destroy cancer cells under the effect of photodynamic process.^[3] The main advantages of this therapy include its non-invasive or minimally invasive nature, selective effect on cancer cells, lack of toxic side effects and lack of drug resistance, and this approach can be used in the treatment of OSCC. Photosensitizer, laser irradiation and oxygen play an important role in the photodynamic process. The possible mechanism is that the photosensitizer leads to the generation of energy through specific laser irradiation and the mobilization of surrounding oxygen molecules, resulting in the formation of reactive oxygen species. Reactive oxygen species leads to oxidative damage of cancer cells and induce apoptosis or necrosis of cancer cells.^[4] 5-aminolevulinic acid (5-ALA) is the second-generation photosensitizer and an endogenous 5-carbon compound, which can produce strong photosensitive protoporphyrin IX. It can have photodynamic effect after laser irradiation. 5-ALA-mediated photodynamic therapy can specifically kill cells or cancer cells that proliferate too fast in the tissue, without affecting normal tissues. Given that protoporphyrin IX has quick metabolism, there is no need to avoid light for a long time after treatment.^[5] The current study was conducted to observe the effect of 5-ALA-mediated photodynamic therapy on OSCC in vitro, with an attempt to explore its underlying role and mechanism.

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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^#