氨基酮戊酸光动力疗法对鳞状细胞癌细胞株A431蛋白激酶D1及其磷酸化位点的影响

目的 研究氨基酮戊酸光动力疗法（ALA-PDT）对皮肤鳞状细胞癌A431细胞蛋白激酶D1（PKD1）的影响，探讨ALA-PDT诱导A431细胞凋亡的机制。方法 体外培养A431细胞，用CCK-8法检测筛选出抑制作用最强的ALA浓度和光动力照射（PDT）剂量组合。将对数期生长A431细胞随机分为不予任何干预的对照组、ALA组、PDT组及ALA-PDT组，观察4组细胞接受相应干预12、24、36、48 h后细胞增殖抑制率和增殖抑制作用最强时间点的凋亡率。反转录PCR检测ALA-PDT作用A431细胞不同时间点后各组PKD1基因mRNA的表达。Western 印迹法检测各组A431细胞中PKD1及其磷酸化位点Tyr463蛋白（pTyr463）、Ser916蛋白（pSer916）表达。结果 ALA浓度1.5 mmol/L、光照剂量2 J/cm2为最佳剂量组合。4组细胞干预12、24、36、48 h的增殖抑制率差异有统计学意义（F = 39.56，P < 0.05）。孵育24 h时：ALA-PDT组细胞增殖抑制率（46.26% ± 1.25%）高于ALA组（14.65% ± 0.33%）、PDT组（14.96% ± 0.68%）和对照组（11.98% ± 0.32%），差异有统计学意义（均P < 0.05 ）；ALA-PDT组细胞增殖抑制率高于12 h（P < 0.05）；4组细胞凋亡率差异有统计学意义（F = 16.32，P < 0.05），ALA-PDT组（41.92% ± 3.23%）高于对照组（4.67% ± 0.88%）、ALA组（7.02% ± 1.52%）和PDT组（8.37% ± 0.59%），均P < 0.05。ALA-PDT作用于A431细胞后0、6、12、24、36、48 h，细胞PKD1基因mRNA的相对表达量差异有统计学意义（F = 22.24，P < 0.05），孵育24 h mRNA相对表达量低于0、6、12 h（均P < 0.05），与36、48 h差异无统计学意义（均P > 0.05）。4组A431细胞中pSer916表达量差异无统计学意义（F = 1.53，P > 0.05），PKD1和pTyr463表达量差异有统计学意义（F值为10.04、8.27，均P < 0.05），ALA-PDT组PKD1、pTyr463的表达低于对照组、ALA组、PDT组（均P < 0.05）。结论 PKD1可能参与ALA-PDT疗法诱导A431细胞凋亡的光化学反应进程，且可能是通过Tyr463位点活化促进癌变的发展。

Effect of aminolevulinic acid-based photodynamic therapy on the of protein kinase D1 and its phosphorylation sites in a cutaneous squamous cell carcinoma cell line A431

Gu Jing, Wang Fuliang, Wang Laiqun, Liu Baoguo, Zhou Meng, Miao Guoying, Li Xiaojing Department of Dermatology, Henan Hongli Hospital, Changyuan 453400, Henan, China （Gu J, Wang LQ）; Graduate School of Hebei Medical University, Shijiazhuang 05000, China （Wang FL）; Department of Dermatology, Affiliated Hospital of Hebei University of Engineering, Handan 056002, Hebei, China（Liu BG, Zhou M, Miao GY, Li XJ） Corresponding author: Liu Baoguo, Email: lbg66@163.com 【Abstract】 Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy （ALA-PDT） on the of protein kinase D1 （PKD1） in a cutaneous squamous cell carcinoma cell line A431, and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 cells. Methods A431 cells were cultured in vitro, and cell counting kit-8 （CCK-8） assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect. A431 cells at exponential growth phase were randomly divided into 4 groups: control group receiving no treatment, ALA group treated with ALA solution alone, PDT group treated with PDT alone, and ALA-PDT group treated firstly with ALA solution and then with PDT. After 12-, 24-, 36- and 48-hour additional culture, CCK-8 assay was conducted to evaluate the cellular proliferation inhibition, and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry. RT-PCR was performed to determine the of protein kinase D1 gene （PRKD1） in A431 cells at different time points after the ALA-PDT treatment, and Western blot analysis to measure protein of PKD1 and its phosphorylation at Tyr463 （pTyr463） and Ser916 （pSer916） in A431 cells. Results The combi-nation of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect. After 12-, 24-, 36- and 48-hour additional culture, there were significant differences in the proliferation inhibition rate among the 4 groups （F = 39.56, P < 0.05）. At 24 hours after the treatment, the ALA-PDT group showed significantly higher proliferation inhibition rate （46.26% ± 1.25%） compared with the ALA group （14.65% ± 0.33%, P < 0.05）, PDT group （14.96% ± 0.68%, P < 0.05） and control group （11.98% ± 0.32%, P < 0.05）, as well as compared with that at 12 hours （P < 0.05）. At 24 hours after the treatment, the apoptosis rate significantly differed among the 4 groups （F = 16.32, P < 0.05）, and the ALA-PDT group showed a significantly higher apoptosis rate （41.92% ± 3.23%） compared with the control group （4.67% ± 0.88%, P < 0.05）, ALA group （7.02% ± 1.52%, P < 0.05） and PDT group （8.37% ± 0.59%, P < 0.05）. At 0, 6, 12, 24, 36 and 48 hours after the treatment, there were significant differences in the mRNA of PRKD1 among the 4 groups （F = 22.24, P < 0.05）, and the mRNA of PRKD1 at 24 hours was significantly lower than that at 0, 6, 12 hours （all P < 0.05）, but was not significantly different from that at 36 and 48 hours （both P > 0.05）. No significant difference in the Ser916-phosphorylated PKD1 was found among the 4 groups （F = 1.53, P > 0.05）, while there were significant differences in the of PKD1 and Tyr463-phosphorylated PKD1 among the 4 groups （F = 10.04, 8.27, both P < 0.05）. Additionally, the ALA-PDT group showed significantly lower of PKD1 and Tyr463-phosphorylated PKD1 compared with the control group, ALA group and PDT group （all P < 0.05）. Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT, and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation.