蛋白激酶C活性、表达及亚细胞分布与KBV200细胞多药耐药相关性的研究(英文)

Correlation of protein kinase C isoforms expression and multidrug resistance in KBV200 cells

目的研究多药耐药肿瘤细胞PKC活性、PKC亚型的表达和亚细胞分布与KBV200细胞多药耐药的关系。方法MTT法检测敏感株KB和耐药株KBV200细胞的耐药性;32P掺入法测定PKC的活性;Westernblot法检测KBV200及其亲本KB细胞株PKC亚型的表达和亚细胞分布。结果长春新碱(VCR)和阿霉素(ADR)对KBV200细胞的IC50值分别高于KB细胞(P<0.01);耐药指数(RI)分别为65.03和19.8。KBV200细胞的膜组分、浆组分的PKC活性均较KB细胞高,KBV200细胞的总PKC活性是KB细胞的2.12倍。Westernblot结果发现KBV200和KB细胞膜组分及浆组分均有PKCα的表达,且KBV200细胞的表达较KB明显增强。PMA可升高KBV200细胞的PKC总活性和膜组分PKC活性,降低浆组分PKC活性、表达(P<0.01);PMA可升高VCR、ADR对KBV200细胞的IC50值(P<0.01)。SP可降低PKC活性;SP预孵育使PKCα膜组分和浆组分的表达均降低,可降低VCR、ADR对KBV200细胞的IC50值(P<0.01)。结论KBV200细胞的PKC活性、表达、亚细胞分布与KB细胞有明显差别,这种差别可能与KBV200耐药性的变化密切相关,在PKC亚型中,PKCα的表达明显高于其他亚型,且高于亲本株,提示KBV200细胞中PKCα与多药耐药相关。

[Objective] To investigate the relationship between protein kinase C （PKC） isoforms and multidrug resistance （MDR） in KBV200 cells. [Methods] The cell inhibitory rate was evaluated by MTT. Western blot was used for the assessment of PKC isoforms expression and subcellalar distribution. PKC activity was assayed by the measurement of the incorporation of ^32P from [γ-^32P]ATP into peptide substrates. [Results] The values of ICS0 of vincristine （VCR） and adriamycin （ADR） in KBV200 ceils were higher than those in KB ceils. The resistance indexes of VCR and ADR were 65.03 and 19.8 respectively. The expression of PKCα was significantly increased in KBV200 cells as compared with parental KB ceils, but PKCβ and s expression in KBV200 ceils had no significant difference from those in KB cells. PKC activity of membrane and cytosol fraction was higher in KBV200 cells than that in KB ceils. Total PKC activity in KBV200 ceils was 1.12-fold higher than that in KB ceils. The PKC promoter PMA had positive effects on PKC activity and expression and the PKC inhibitor SP had negative effect in KBV200 ceils. PMA increased MDR and SP decreased MDR in KBV200 ceils. [Conclusion] PKC isoforms expression and subcellular distribution in KBV200 ceils were different significantly from those in KB ceils. The alteration of PKC activity,expression and subcellular distribution might be related to the change of resistance in KBV200 cells. Of the isoforms detected, PKCα might be the most important one to MDR in KBV200 ceils.