两种关键信号转导通路活性状态与K562细胞对DNR、Ara-C敏感性的关系

Research for sensitivity of K562 cell line to the chemotherapeutic drugs under two key signal transduction pathways of different activity status

目的观察两种关键信号转导通路的活性状态与白血病细胞株K562对柔红霉素(DNR)、阿糖胞苷(Ara-C)的敏感性的关系。方法将K562细胞分为5个组,经U0126、LY294002、雷帕霉素处理的细胞分别为抑制剂A、B、C组,经佛波酯(PMA)刺激的细胞为激活剂组,未作处理者为对照组。采用磷酸化流式细胞术检测细胞中的p-Akt T308、p-Akt S473及p-ERK1/2。将各组细胞分别与DNR、Ara-C作用48 h,采用AnnexinⅤ/PI标记法检测细胞凋亡率。结果抑制剂A组p-ERK1/2表达及抑制剂B组p-ERK1/2、p-Akt T308、p-Akt S473表达均低于对照组(P均<0.05);激活剂组p-ERK1/2、p-Akt S473表达高于对照组(P均<0.05);抑制剂C组p-ERK1/2、p-AktT308、p-Akt S473表达及激活剂组p-Akt T308与对照组相近(P均>0.05)。DNR和Ara-C作用后激活剂组细胞凋亡率均低于对照组(P均<0.05)。DNR作用后,抑制剂A、B、C组细胞凋亡率均高于对照组(P均<0.05);Ara-C作用后,抑制剂B、C组的细胞凋亡率高于对照组(P均<0.05),抑制剂A组与对照组凋亡率无显著差异(P>0.05)。结论在信号转导通路激活状态下,K562对DNR、Ara-C的敏感性降低,抑制Ras/PI3K/PTEN/Akt/mTOR通路能增强K562对DNR、Ara-C的敏感性,Ras/Raf/MEK/ERK通路激活可能与DNR耐药有关,但抑制该通路对Ara-C诱导的K562细胞凋亡无直接作用。

Objective To observe the sensivity of K562 cell lines to DNR and Ara-C under two key signal transduction pathways of different activity status. Methods K562 cells were divided into five groups. Inhibitor groups A, B, C were treated with U0126, LY294002, rapamycin, respectively. Activator group was treated with phorbol （PMA）. Cells without any stimulate considered as control group. Adapt phospho-specific flow cytometry to analyse the expression of Akt T308 ,Akt $473 and ERK1/2. Each group of cells incubated with DNR and Ara-C respectively for 48 hours. The apoptosis rate of K562 cells were detected by Annexin V/PI. Expression of p-ERK1/2 of group A and p-ERK1/2 ,p-Akt T308, p-Akt $473 of group B were lower than control group（ all P 〈 0.05 ）. The expression of p-ERK1/2, p-Akt $473 of activator group were higher than control group（ all P 〈 0.05 ）. The expression of p-ERK1/2, p-Akt T308, p-Akt $473 of group C and p-Akt T308 of activator group were close to control group（ all P 〉 0.05 ）. The apoptosis rate of activator group induced by DNR and Ara-C were lower than control group （all P 〉 0.05 ）. The apoptosis rate of group A, B, C induced by DNR were higher than control group（ all P 〈 0.05 ）. The apoptosis rate of group B, C induced by Ara-C were higher than control group（ all P 〈 0.05 ）. The apoptosis rate of group A induced by Ara-C has no different with control group（ P 〉 0.05 ）. Conclutions The activate of signal transduction pathways can reduce the sensitivity of K562 cells to DNR and Ara-C. Ras/Raf/MEK/ ERK pathways may correlated with resistanceto DNR, but inhibit this pathways has no direct relation to the apoptosis in- duced by Ara-C.