Zhu M Q, Shi S R, Wan G Y, Wang Y S, Wang Y, Zhang L Y, Zhao Y H
Department of Orthodontics, Stomatological Hospital, Tianjin Medical University, Tianjin 300070, China.
Department of Prosthodontics, Stomatological Hospital, Tianjin Medical University, Tianjin 300070, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2019 Mar 9;54(3):176-182. doi: 10.3760/cma.j.issn.1002-0098.2019.03.006.
To investigate the and effects of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy against oral squamous cell carcinoma (OSCC) and preliminarily explore the possible mechanisms. 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. 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)%, 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), 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 (0.05). 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 and .
探讨5-氨基酮戊酸（5-ALA）介导的光动力疗法对口腔鳞状细胞癌（OSCC）的作用及机制。将SCC25细胞分为对照组（5-ALA浓度为0 mg/L）和实验组（5-ALA浓度分别为10、25、50、100和150 mg/L）。采用流式细胞术检测5-ALA诱导SCC25细胞内原卟啉Ⅸ（PpⅨ）的生成。将SCC25细胞分为对照组（5-ALA浓度为0 mg/L）、单纯激光组、单纯5-ALA组（5-ALA浓度为100 mg/L）和5-ALA联合激光照射组（5-ALA浓度分别为5、10、25、50和100 mg/L），采用甲基噻唑基四氮唑法评估5-ALA联合激光照射（波长635 nm，功率密度87 mW/cm²，激光剂量10.4 J/cm²）对SCC25细胞的细胞毒性（每组孵育时间分别为4、8和12 h），并通过流式细胞术评估联合治疗对细胞凋亡的诱导作用（每组孵育时间为12 h）。采用荧光探针法测定5-ALA联合激光照射诱导的细胞内活性氧（ROS）生成（每组孵育时间为12 h）。建立荷SCC25肿瘤的小鼠OSCC异种移植模型，将小鼠分为对照组（生理盐水）、5-ALA组（5-ALA浓度为50 mg/kg）和5-ALA联合激光照射组（5-ALA浓度分别为10、25和50 mg/kg）。进一步检测5-ALA联合激光照射（波长635 nm，功率密度158 mW/cm²，激光剂量94.8 J/cm²）的抗肿瘤作用。5-ALA诱导SCC25细胞内PpⅨ的生成呈药物浓度（0～150 mg/L）和孵育时间（0～24 h）依赖性。当5-ALA浓度为100 mg/L时，细胞内PpⅨ生成处于相对稳定状态。5、10、25、50、100 mg/L 5-ALA联合激光照射后的细胞活力和凋亡率分别为（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）%，与对照组[（96.3±6.0）%，（0.35±0.13）%，P＜0.05]相比，5-ALA联合激光照射显著抑制SCC25细胞生长并诱导细胞凋亡。联合治疗（5、10、25、50和100 mg/L 5-ALA联合激光照射）后，二氯荧光素平均荧光强度分别为（1.46±0.12）×10⁴、（2.16±0.30）×10⁴、（3.57±0.34）×10⁴、（81.70±13.05）×10⁴、（113.00±7.35）×10⁴，与对照组[（0.96±0.15）×10⁴，P＜0.05]相比，SCC25细胞内产生大量ROS，且与细胞内PpⅨ含量呈正相关。与对照组相比，5-ALA（浓度为10、25和50 mg/kg）联合激光照射显著抑制荷SCC25肿瘤小鼠的肿瘤生长（P＜0.05）。5-ALA介导的光动力疗法可触发细胞内ROS生成，具有显著的细胞毒性和凋亡诱导作用，从而体内外抑制肿瘤生长。
