锦鸡丙素对两株人肺癌细胞株蛋白激酶和同工酶活性的影响

Effects of Miyabenol-C on Protein Kinase-C in Two Lung Carcinoma Cell Lines

目的 研究锦鸡丙素对两种人肺癌细胞株中不同组分的蛋白激酶C(PKC)活性及其同工酶分布的影响 ,探讨两种细胞株对锦鸡丙素的细胞生长抑制作用表现出敏感性差异的原因。方法 梯度离心法、32 P掺入法及蛋白印迹法。结果 ①A5 4 9及HCI H4 46细胞所有组分中的PKC活性均可被锦鸡丙素所抑制 ,两种细胞胞质及颗粒组分中PKC活性完全受抑所需时间相同 ,A5 4 9细胞全细胞组分及核组分所需时间均长于NCI H4 46细胞 ;②A5 4 9细胞胞质组分中PKC的活性不可逆转 ,而HCI H4 46细胞为核组分 ;③ 5 4 9细胞中主要表达PKC α、ε和ζ ,胞质中三者都存在 ,颗粒组分中只检测到α且锦鸡丙素处理后α消失 ,而胞质及全细胞组分中的α含量增加 ;NCI H4 46细胞胞质中主要存在PKC ζ和ε,颗粒组分中仅检测到PKC ζ且锦鸡丙素处理后胞质中 ζ增加而颗粒组分中则减少。结论 锦鸡丙素体内外均可抑制PKC的活性并可降低PKC α在颗粒组分中的含量使其膜转位受阻 ,但这种抑制作用与肿瘤细胞生长的抑制之间并没有直接的联系 ;PKC α可能是A5 4 9细胞信号传导系统中最主要的PKC同工酶形式 ,锦鸡丙素抑制其活性并阻碍其膜转位将可能影响A5 4 9肺癌细胞的增殖并与A5 4 9细胞对锦鸡丙素敏感性较高有着密切的关系。

Purpose To study the relationship between altered susceptibilities of A549 and NCI?H446 cell lines to miyabenol c and the inhibitory effect of this compound on PKC isoenzymes. Methods Total cell,cytosolic and particulate fraction were extracted by graded centrifugation and nuclear fraction was extracted by sucrose graded centrifugation.PKC activity was evaluated by means of incorporation of isotope 32 P and six PKC isoforms α,β,γ,δ,ε,ζ were detected by western blotting in four cell fractions of A549 and NCI?H446 cells. Results PKC activity of two cell lines could be inhibited thoroughly after 2?h treatment with 20?μmol/L miyabenol C for cytosolic fractions,but it only required 2?min for particulate fractions.Total loss of activity required 5?min for nuclear fraction in NCI?H446 cells but it required 10?min in A549 cells.More than 90?min was needed to inhibit PKC activity entirely in A549 than in NCI?H446 nuclear fraction.PKC?α could be detected by western blotting in cytosolic and particulate fractions of A549 cells accompanied by slightly increase in cytosolic fraction and disappearance in particulate fraction after treatment with the drug.PKC?ε and PKC? ζ also could be detected in A549 cytosolic fraction but not in particular fraction.PKC?ε and PKC?ζ existed in NCI?H446 cytosolic fraction;the former decreased in particulate fraction whereas the latter increased in cytosolic fraction after drug treatment. Results PKC activity of two cell lines could be inhibited thoroughly after 2?h treatment with 20?μmol/L miyabenol C for cytosolic fractions,but it only required 2?min for particulate fractions.Total loss of activity required 5?min for nuclear fraction in NCI?H446 cells but it required 10?min in A549 cells.More than 90?min was needed to inhibit PKC activity entirely in A549 than in NCI?H446 nuclear fraction.PKC?α could be detected by western blotting in cytosolic and particulate fractions of A549 cells accompanied by slightly increase in cytosolic fraction and disappearance in particulate fraction after treatment with the drug.PKC?ε and PKC? ζ also could be detected in A549 cytosolic fraction but not in particular fraction.PKC?ε and PKC?ζ existed in NCI?H446 cytosolic fraction;the former decreased in particulate fraction whereas the latter increased in cytosolic fraction after drug treatment. Conclusions It can be concluded that miyabenol c are able to inhibit PKC activity in vitro and in vivo and decrease content of PKC isoenzymes in particulate fraction.The results suggest that it can interfere membrane translocation of PKC,but there is no direct relationship between inhibition of PKC and the growth arrest of tumor cells as well as different susceptibility to miyabenol C in two cell lines.PCK?α might be the principle isotype expressed in A549 cells which plays a very important role in signal transduction,growth and high susceptibility to miyabenol C of A549 lung carcinoma cells.