蛋白激酶C激活剂和抑制剂对肿瘤坏死因子杀瘤活性的影响

以小鼠成纤维细胞瘤L929细胞为靶细胞,研究蛋白激酶C(PKC)的激活剂和抑制剂对人重组肿瘤坏死因子(rHuTNF)杀瘤活性的影响.各种药物与rHuTNF 10 ng·ml~(-1)和放线菌素D 1 μg·ml~(-1)温育18 h,结果表明PKC的激活剂佛波醇-12-肉豆蔻酸盐-13-乙酸盐(PMA)2.5～160ng·ml~(-1)可浓度依赖性地抑制rHuTNF的杀瘤活性.Sc-10(1～16μg·ml~(-1))单独作用很弱,但可浓度依赖地增强PMA 10 ng·ml~(-1)或A23187 0.5μg·ml~(-1)的抑制作用,PKC抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪(H-7)单独作用对rHuTNF杀瘤活性无影响,但可减弱PMA 50ng·ml~(-1)的抑制作用.槲皮囊2～16μg·ml~(-1)则可直接抑制rHuTNF的杀瘤活性,钙调蛋白抑制剂N-(6-氨己基)-5-氯-1-萘磺酰胺(W-7)及其同系物也有微弱的抑制作用.结果提示PKC在rHuTNF杀瘤作用中起着重要作用.

Effects of protein kinase C activators and inhibitors on tumor necrosis factor cytotoxicity

A variety of protein kinase C(PKC)activators and inhibitors were tested for their ability to interfere with mouse fibroblast L929 cells lysis.Compounds were added simultaneously with recombinant human tumor necrosis factor(rHuTNF,10 ng·ml-1)in the presence of actinomycin D 1μg·.ml-1 and the cytotoxicity was measured after 18 h by means of the crystal violet staining assay.Purified rHuTNF of a concentration of 10 ng·ml-1 produced about 93% cytotoxicity.PKC activator phorbol 12-myristate 13-acetate(PMA)had an inhibitory activity in a dose dependent manner; another activator N-(n-heptyl)-5-chloro-l-naphthalenesulfonamide(Sc-10)showed a weak inhibitory effect,but significantly inhibited TNF-mediated L929 lysis in combination with PMA10 ng·ml-1 or calcium ionophore A23187 0.5μg·ml-1 PKC inhibitor 1-(5-isoquino-linesulfonyl)-2-methylpiperazine(H-7)had no inhibitory effect on TNF cytotoxicity but could reduce the inhibitory activity of PMA 50ng·ml-1 whereas PKC inhibitor quercetin markedly inhibited TNF cytotoxicity.Calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide(W-7)and its homologs exhibited a weak inhibitory activity.These results suggest PKC plays an important role in TNF mediated cytotoxicity.