Effect and mechanism of palmatine hydrochloride mediated laser therapy on proliferation and apoptosis in oral squamous cell carcinoma CAL-17 cells
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摘要:
背景 口腔鳞状细胞癌主要的治疗方法为手术、放疗和化疗等，总体预后不尽如人意。近年来光动力疗法(photodynamic therapy，PDT)凭借自身的优势广泛应用口腔癌治疗，其中光敏剂至关重要。因此，选择安全有效的光敏剂是取得良好光动力疗效的关键。 目的 研究盐酸巴马汀(palmatine hydrochloride，PaH)联合激光(Laser)对口腔鳞状细胞癌(oral squamous cell carcinoma，OSCC)细胞株CAL-27生长、凋亡的影响及其机制。 方法 将CAL-27细胞随机分为对照组、盐酸巴马汀(PaH)组(0 ～ 16 μmol/L)、激光(Laser)组(0 ～ 2.4 J/cm2)及盐酸巴马汀联合激光(PaH-PDT)组(PaH 2 μmol/L + Laser 2.4 J/cm2)，CCK-8法检测各组细胞的生长情况，流式细胞术检测各组细胞周期分布及凋亡率，DCFH-DA探针检测细胞内活性氧(reactive oxygen species，ROS)生成，Western blot法检测各组细胞周期及凋亡相关蛋白的表达。 结果 与对照组比较，PaH组和Laser组CAL-27细胞增殖无统计学差异，PaH-PDT组CAL-27细胞增殖明显抑制，且呈药物浓度-激光强度依赖关系。在激光强度分别为1.2 J/cm2、1.8 J/cm2和2.4 J/cm2时，PaH的半数抑制浓度(IC50)分别为8.3 μmol/L、4.1 μmol/L和2.1 μmol/L。PaH-PDT诱导CAL-27细胞发生G2/M期阻滞，下调Cyclin B1和CDK1表达，上调P21和P27表达。PaH-PDT作用CAL-27细胞后，细胞凋亡率由3.4%±0.7%上升至35.17%±3.9%(P＜0.05)。PaH-PDT诱导细胞内ROS生成，上调P53、Bax的表达，下调Bcl-2的表达。 结论 PaH可以作为一种新型光敏剂介导光动力疗法抑制口腔癌细胞CAL-27增殖，诱导其凋亡，其机制可能与PaH-PDT诱导细胞G2/M期阻滞和调控凋亡蛋白P53、Bax和Bcl-2的表达有关。 Abstract:Background The available treatments for oral squamous cell carcinoma mainly include surgery, radiotherapy, chemotherapy and so on, but the overall prognosis is still not satisfactory. Photodynamic therapy has been widely used in the treatment of oral cancers by its own advantages in recent years, and photosensitizer is the most important point. Therefore, the selection of a safe and effective photosensitizer is the key to achieve good results of photodynamic therapy. Objective To investigate the effects of Palmatine hydrochloride (PaH) combined laser on the growth and apoptosis of oral squamous cell carcinoma (OSCC) cell CAL-27 and its mechanism. Methods CAL-27 cells were randomly divided into control group, PaH group, Laser group and PaH combined with Photodynamic therapy (PaH-PDT) group (PaH: 2 μM + Laser: 2.4 J/cm2). CCK-8 assay was used to detect the viability of cells. Flow cytometry was used to detect each cell cycle distribution and apoptosis rate. DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS) generation, and western blot was used to detect the expression of cell cycle and apoptosis related proteins. Results Compared with control group, PaH and laser alone had no significant effect on the growth of CAL-27 cells, while PaH-PDT significantly inhibited cell proliferation. When the laser intensity was 1.2 J/cm2, 1.8 J/cm2 and 2.4 J/cm2, the IC50 of PaH was 8.3 μmol/L, 4.1 μmol/L and 2.1 μmol/L, respectively. PaH-PDT increased the ratio of cells in the G2/M phase, down-regulated the expressions of Cyclin B1 and CDK1, and up-regulated the expressions of P21 and P27. After treated by PaH-PDT, the apoptosis rate increased from (3.4±0.7)% to (35.17±3.9)% (P＜0.05). PaH-PDT induced intracellular ROS production, up-regulated the expression of P53 and Bax, and down-regulated Bcl-2. Reactive oxygen scavengers showed that NAC could significantly reverse the effect of PaH-PDT on apoptotic proteins.中文摘要中没看到这句 Conclusion aaPAH can be used as a new photosensitizing agent to mediate the photodynamic therapy to inhibit the proliferation of oral cancer cell CAL-27 and induce its apoptosis. The mechanism may be related to the G2/M phase arrest induced by PaH-PDT and ROS production involved in the regulation of apoptotic proteins. -
图 1 CCK-8法检测CAL-27细胞活力 (aP < 0.05, vs 0 μmol/L; bP < 0.01, vs 0 J/cm2)
Figure 1. Viability of CAL-27 cells tested by CCK-8 assay (aP < 0.05, vs 0 μmol/L; bP < 0.01, vs 0 J/cm2)
图 2 流式细胞术检测CAL-27细胞凋亡(aP < 0.05，vs 对照组)
Figure 2. Apoptosis of CAL-27 cells detected by flow cytometry (aP < 0.05, vs control)
图 3 CAL-27细胞周期分析
A：流式细胞术分析对照组细胞周期分布百分比；B：流式细胞术分析PaH-PDT组细胞周期分布百分比；C：柱状图比较两组不同周期细胞百分比(aP < 0.05，vs 对照组)
Figure 3. Cell cycle analysis of CAL-27 cells
A: the percentages of cell cycle distribution of control group were analyzed using flow cytometry; B: the percentages of cell cycle distribution of PaH-PDT group were analyzed using flow cytometry; C: histogram represents the percentage of cells arrested in different phases of cell cycle (aP < 0.05, vs control)
图 4 Western blot检测CDK1、Cyclin B1、P21、P27的表达水平
Figure 4. Expression level of Cyclin B1, CDK1, P21 and P27 determined by western blot. β-actin served as loading control
图 5 DCFH-DA标记细胞，荧光显微镜观察(400×)，PaH-PDT诱导CAL-27细胞内ROS生成
Figure 5. The cells were labeled with DCFH-DA and observed by fluorescence microscopy (400×), PaH-PDT induced ROS production in CAL-27 cells
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