Abstract:

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.