抑制或沉默Akt基因表达可逆转人结肠癌耐药SW1116/HCPT细胞的耐药性

Reversal effect of inhibiting or silencing the expression of Akt gene on hydroxycampothecin-resistance of colorectal cancer SW1116/HCPT cells

目的 :研究阻断磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/丝氨酸-苏氨酸激酶(seride-threonine protein kinase,又称Akt)信号通路对逆转耐羟基喜树碱(hydroxycamptothecin,HCPT)人结肠癌SW1116/HCPT细胞多药耐药性的影响。方法:采用蛋白质印迹法检测亲本SW1116细胞和耐HCPT细胞株SW1116/HCPT中Akt及磷酸化Akt(phospho-Akt,p-Akt)蛋白的表达水平。应用针对PI3K/Akt信号通路的小分子抑制剂LY294002或siR NA干扰的方法分别抑制或沉默Akt蛋白的表达;MTT法检测抑制或沉默Akt蛋白表达后,HCPT对SW1116/HCPT细胞生存率的影响;实时荧光定量PCR及蛋白质印迹法检测对耐药转运体三磷酸腺苷结合盒转运体G(2ATP-binding cassette transporter G2,ABCG2)mR NA及蛋白表达的影响;罗丹明123(rhodamine123,Rh123)法检测对SW1116/HCPT细胞药物外排功能的影响。结果 :蛋白质印迹法检测结果发现,耐药SW1116/HCPT细胞中p-Akt的表达水平明显高于亲本SW1116细胞(P<0.01)。小分子抑制剂LY294002及靶向Akt基因的Akt-siR NA均能明显降低p-Akt的表达水平,增加细胞对HCPT的药物敏感性(P值均<0.01),有明显的多药耐药逆转作用。LY294002作用48 h可使耐药转运体ABCG2 mR NA的表达水平下调(74.82±4.71)%,蛋白表达水平下调(58.24±4.78)%(P值均<0.01),并且耐药蛋白体的转运功能也明显受到抑制,细胞内Rh123的浓度增加了(1.45±0.12)倍(P<0.01)。沉默Akt基因后ABCG2 mR NA的表达水平下降了(59.63±5.14)%,蛋白表达水平下降了(44.41±2.56)%(P值均<0.01),细胞内Rh123的浓度增加了(1.22±0.10)倍(P<0.01)。结论:PI3K/Akt信号通路可正向调节耐药基因ABCG2的表达,在调节HCPT诱导的多药耐药过程中发挥重要作用。

Objective： To investigate the reversal effect of blocking phosphatidylinositol 3-kinase （PI3K）/ protein kinase B （Akt） signal pathway on hydroxycampothecin （HCPT）-resistance of colorectal cancer SWl 116/HCPT cells. Methods： The expression levels of Akt and phospho-Akt （p-Akt） in the parent cell line SWl 116 and HCPT-resistant cell line SWl 116/HCPT were detected by Western blotting. The specific inhibitor LY294002 and siRNA targeting Akt gene were used to block the expression of Akt. Then the proliferation of SW1116/HCPT cells was detected by MTT assay. The expression levels of ATP-binding cassette transporter G2 （ABCG2） mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The drug-efflux function was detected by Rhodamine 123 （Rh123） method. Results： The expression level of p-Akt in SWl 116/HCPT cells was higher than that in parent SWl 116 cells （P 〈 0.01）. LY294002 and Akt-siRNA could inhibit the expression level of p-Akt, suppress the proliferation of SWl 116/HCPT cells, and increase the sensitivity to HCPT （all P 〈 0.01）. LY294002 could down-regulate the expressions of ABCG2 mRNA and protein by （74.82±4.71）% and （58.24±4.78）% （both P 〈 0.01）, respectively. The accumulation of Rh123 in SWl 116/HCPT cells was increased 1.45±0.12 times 48 h after treatment （P 〈 0.01）. After silencing the expression of Akt, the expressions of ABCG2 mRNA and protein were decreased by （59.63±5.14）% and （44.41 ±2.56）% （both P 〈 0.01）, respectively, and the concentration of intracellular Rh123 was increased 1.22±0.10 times （P 〈 0.01）. Conclusion： PI3K/Akt signal pathway can up-regulate the expression of drug-resistance gene ABCG2, and play a vital role in the carcinogenesis of multidrug-resistance induced by HCPT.