获得性顺铂耐药的大肠腺癌LS-174T细胞核中YB-1的活性改变及其对P-gp和PCNA转录表达的影响

Increased nuclear localization of transcription factor YB-1 and its subsequent influence on transcription of P-gp and PCNA in acquired cisplatin-resistant colon adenocarcinoma cell line

目的:观察耐药细胞株LS-174T核中YB-1的活性改变及其对P-gp和PCNA转录表达水平的影响,探讨YB-1在肿瘤顺铂耐药机制中的作用。方法:采用顺铂药物浓度递增法、间歇性作用的体外诱导法建立人结肠癌的多重耐药细胞系LS-174T;用EMSA检测耐药肿瘤细胞核中转录因子YB-1活性的改变,半定量RT-PCR检测P-gp和PCNA mRNA表达水平的改变。结果:耐药细胞株核中YB-1的活性明显增加,且P-gp mRNA和PCNA mRNA的表达水平均较非耐药细胞株明显增高(P<0.01)。结论:顺铂耐药细胞株细胞核内YB-1活性增高促进了P-gp和PCNA转录表达水平,它们可能共同参与顺铂导致DNA损伤的修复,在肿瘤耐药机制中发挥重要作用。

Objective ：To observe the level of YB-1 activity in the nuclei of acquired cisplatin-resistant colon ad cell line and its secondary effect on the transcription of P-gp and PCNA, and to elucidate the association between nuclear YB-1 and cisplatin resistance in human colon adenocarcinoma. Methods：A LS-174T cell line resistant to cisplatin was successfully established by exposing cells to increasing cisplatin concentrations ranging from 200 ng/ml to 2 000 ng/ml on an intermittent dosage schedule, Intranuclear YB-1 transcriptional activity was examined by EMSA analysis for both cisplatin sensitive and resistant human colon adenocarcinoma cell line. The alterations of P-gp and PCNA mRNA levels were measured by semi-quantitative RT-PCR. Results：EMSA analysis for nuclear extracts indicated that cisplatin-resistant cells showed much higher nuclear YB-1 activity than that of sensitive parental cells. Meanwhile, the transcriptional expression of P-gp and PCNA gene was also increased as well （P 〈 0. 01 ）. Conclusion：The increased activity of YB-1 in the nuclei enhanced the P-gp and PCNA gene expression at transcriptional level. They might be involved in DNA repair in LS-174T cells and play an important role in the drug resistance mechanism.