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

Abstract

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression 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 expression 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 expression 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) . No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F= 1.53, P > 0.05) , while there were significant differences in the expression 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 expression 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. Key words: Photochemotherapy; Aminolevulinic acid; Neoplasms, Squamous Cell; Protein kinases; Primary cell culture; A431 cell