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目的 探讨半边旗提取物 6F的细胞毒作用及诱导DNA片段化与蛋白激酶C(PKC)信号转导途径的关系 ,检测 6F对PKC活性的影响。方法 受试对象为HL 6 0细胞 ,超速离心法获得的胞液 (可溶部分 )及颗粒 (不可溶部分 ,包括细胞膜系统及胞核 )部分用作PKC活性测定。经 0 .4g·L- 1磷脂酰丝氨酸 ,0 .0 4g·L- 1甘油二油酸酯激动剂作用酶粗提物后 ,用液体闪烁计数仪计数 [γ 32 P]ATP参入外源底物的量以测定PKC活性。MTT法测定HL 6 0细胞的活力 ,二苯胺法测定 6F诱导DNA片段化程度。结果 在所测试的浓度范围内 (0 .5～ 312 μmol·L- 1) ,化合物 6F显著抑制胞液及颗粒部分PKC活性 ,最大抑制率达 88.6 % ,呈浓度依赖关系 (胞液部分r =0 .781,P <0 .0 5 ,颗粒部分r =0 .931,P <0 .0 1)。 6F诱导HL 6 0细胞DNA片段化及对细胞的毒性作用可被具有致癌作用的PKC激活剂肉豆蔻酸酯 (PMA ,浓度为 6 5nmol·L- 1)拮抗 ,抑制率分别是 30 %和 4 4% (P <0 .0 1)。PMA单独用使HL 6 0细胞线粒体将MTT还原为甲月赞的能力增强 14 % (P <0 .0 1) ,即增强细胞活力。结论化合物 6F是PKC的抑制剂。 6F对HL 6 0细胞DNA片段化的诱导作用及其细胞毒作用至少可部分归因于其对PKC活性的抑制作用
Objective To investigate the cytotoxicity of 6F from Pteris semipinnata L. and the relationship between DNA fragmentation and PKC signal transduction pathway and to explore the effect of 6F on PKC activity. Methods The target cells were HL 60 cells. The fractions of cytosol (soluble fraction) and particles (insoluble fraction, including membrane system and nucleus) obtained by ultracentrifugation were used as PKC activity assay. After 0.4 g · L-1 phosphatidylserine and 0.04 g · L-1 glycerol dioleate agonist were used as the crude enzyme extracts, [γ 32 P] ATP was counted by a scintillation counter into the exogenous substrate The amount of material was measured for PKC activity. The viability of HL-60 cells was determined by MTT assay and the DNA fragmentation induced by 6F was determined by diphenylamine assay. Results Compound 6F significantly inhibited the PKC activity of cytosolic and granular fractions at the concentration range of 0. 5 ~ 312 μmol·L-1, with the maximum inhibitory rate of 88.6%. In a concentration-dependent manner (cytosol fraction r = 0 .781, P <0. 05, Particle fraction r = 0 .931, P <0.01). 6F-induced DNA fragmentation and cytotoxicity in HL-60 cells were antagonized by the potent PKC activator myristate (PMA at a concentration of 65 nmol·L -1) with inhibitory rates of 30% and 4%, respectively 4% (P <0. 01). PMA alone increased the viability of mitochondria in HL-60 cells by reducing MTT to formazan by 14% (P <0.01), ie enhancing cell viability. Conclusion Compound 6F is an inhibitor of PKC. The induction of DNA fragmentation by 6F in HL 60 cells and their cytotoxicity can be at least partly attributed to their inhibitory effect on PKC activity