银杏酚酸通过抑制PKD-2活性提高Tca8113细胞对化疗药物的敏感性

Ginkgolic acid enhances chemosensitivity of Tca8113 cells via suppressing PKD-2 activity

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 enhanced by ginkgolic acid （GA） in Tca8113 cells. Methods The effect of different concentrations of GA on the proliferation of Tca8113 cells and the cell survival rate for single use of Carboplatin （CBP） or Paclitaxel （PTX） and combination of CBP or trrx with GA was detected by Methyl thiazolyl tetrazolium（MTT） assay. Western blot analysis was used to detect protein expression of phospho-protein kinase D （p-PKD-2） and PKD- 2 in Tea8113 cells induced by GA for different times, shRNA was introduced to downregulate the expression of PKD-2 in Tca8113 cells. Phorbol 12-myristate 13-acetate （PMA） was used to induce the phosphorylation of PKD-2. MTT assay was used to detect viability after exposure of cells to drugs or combination of drugs and GA. Results The 1C50 of GA which was detected by MTI＂ was 20 mg/L. The folds of chemosensitivity of Tca8113 to CBP and PTX enhanced by GA were 4.37 and 39.20 respectively. Exposure of Tea8113 cells to GA resulted in a decrease in phospho-PKD-2 level in a time-dependent manner. Knockdown of PKD-2 with shRNA enhanced chemosensitivity of Tca8113 cells to CBP and PTX or combination with GA, with the folds of 1. 651 7 and 1. 501 6 respectively. GA also antagonized the function of PMA, which induced the phosphorylated activation of PKD2 and increased chemotherapy drugs resistance of Tca8113 cells. Conclusion GA may pro- mote chemosensitivity of TcaSll3 cells via inhibiting phosphorylation of PKD-2 pathway.