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目的探讨蛋白激酶D-2(protein kinase D-2,PKD-2)在银杏酚酸(ginkgolic acid,GA)提高舌鳞状细胞癌细胞株Tca8113对化疗药物敏感性过程中的作用。方法通过MTT方法检测GA对Tca8113的增殖抑制及提高Tca8113对化疗药物敏感性;Western blot检测在GA不同作用时间下PKD-2在Tca8113中的表达及磷酸化特性;利用shRNA干扰技术基因沉默Tca8113中的PKD-2基因;利用佛波酯(PMA)诱导Tca8113中的PKD-2高磷酸化;以MTT方法检测经shRNA干扰后的Tca8113和及经PMA诱导后的Tca8113细胞对GA及化疗药物的敏感性。结果 GA抑制Tca8113生长的半数抑制浓度(IC50)为20 mg/L,GA明显提高Tca8113对化疗药物卡铂(CBP)和紫杉醇(PTX)的敏感性,分别提高4.37倍和39.20倍(P<0.05);在野生型Tca8113中与非磷酸化PKD-2蛋白质表达相比较,GA对Tca8113中磷酸化PKD-2蛋白质的表达呈时间依赖性抑制作用(P<0.05);经shRNA沉默后的Tca8113细胞建立稳定细胞株,同与GA联合用药作用相似,PKD-2基因沉默后的Tca8113细胞对CBP和PTX的敏感性明显提高,分别提高1.651 7倍和1.501 6倍(P<0.05);PMA激活Tca8113中的PKD-2后,Tca8113对CBP和PTX的敏感性明显降低,同时发现GA能明显拮抗PMA作用。结论GA通过抑制PKD-2活性途径提高Tca8113对化疗药物的敏感。
Objective To investigate the role of protein kinase D-2 (PKD-2) in chemosensitivity of tongue squamous cell carcinoma cell line Tca8113 induced by ginkgolic acid (GA). Methods MTT assay was used to detect the proliferation inhibition of Tca8113 and the chemosensitivity of Tca8113 to Tca8113. Western blot was used to detect the expression and phosphorylation of PKD-2 in Tca8113 treated with GA at different time points. (PMA) induced PKD-2 hyperphosphorylation in Tca8113; MTT assay was used to detect the sensitivity of Tca8113 and Pca-induced Tca8113 cells to GA and chemotherapeutic drugs Sex. Results The IC50 of GA3 inhibiting the growth of Tca8113 was 20 mg / L. GA significantly increased the sensitivity of Tca8113 to chemotherapeutic agents carboplatin (CBP) and paclitaxel (PTX) by 4.37 folds and 39.20 folds (P <0.05) ). Compared with non-phosphorylated PKD-2 protein expression in wild-type Tca8113, GA inhibited the phosphorylation of PKD-2 protein in Tca8113 in a time-dependent manner (P <0.05). After shRNA silenced Tca8113 cells The establishment of stable cell lines was similar to that of GA, and the sensitivity of Tca8113 cells after PKD-2 gene silencing to CBP and PTX was significantly increased by 7 times and 1.501 times respectively (P <0.05); PMA activated Tca8113 In PKD-2, Tca8113 significantly decreased the sensitivity of CBP and PTX, also found that GA can significantly antagonize the role of PMA. Conclusion GA can enhance the sensitivity of Tca8113 to chemotherapeutic drugs by inhibiting PKD-2 activity.