吉西他滨耐药胰腺癌细胞系的耐药机制

目的探讨吉西他滨耐药胰腺癌细胞系的耐药机制。方法应用免疫组织化学方法和逆转录PCR法（RT—PCR）,对吉西他滨耐药细胞系的耐药机制进行了研究。选用沙尔威辛（SAL）进行逆转耐药实验,采用了RT—PCR法、流式细胞仪和MTT法,评估SAL逆转耐药的效果。结果免疫组织化学法显示,耐药细胞SW1990-GEM的P-糖蛋白（P—gP）表达为弱阳性,其亲本细胞SW1990为阴性。多药耐药相关蛋白（MRP）在两细胞系表达均为阴性。SW1990-GEM的多药耐药基因（mdr-1）在mRNA水平上的转录比其亲本细胞系SW1990增强;脱氧胞苷激酶（dCK）基因的转录减少;核苷酸还原酶（RR）基因的转录增强。经过SAL作用后,mdr-1在mRNA水平上的转录减少。流式细胞仪显示,经过4,30,60,90nmol／LSAL作用后,细胞内罗丹明123的蓄积累分别增加1．04,1．16,1．39,1．72倍。用4nmol／LSAL进行逆转吉西他滨耐药实验,未接受SAL处理的对照组对吉西他滨的IC50为203．72μmol／L;经SAL作用1h后再加吉西他滨组的IC50为121．36μmol／L（P=0．219）;经SAL作用24h后再加吉西他滨组的IC59为121．64μmol／L（P=0．167）。结论胰腺癌细胞对吉西他滨耐药的原因与dCK减少有关,同时也与mdr-1和RR升高相关。SAL能够下调SW1990-GEM耐药细胞的mdr-1和P—gP表达,但低浓度的SAL不能逆转SW1990-GEM对吉西他滨的耐药。

The mechanism of resistance to 2', 2'-difluorodeoxycytidine (gemcitabine) in a pancreatic cancer cell line

OBJECTIVE: To study the mechanism of resistance and its reversal to 2', 2'-difluorodeoxycytidine (gemcitabine) in a pancreatic cancer cell line SW1990. METHODS: Immunohistochemistry and RT-PCR techniques were used to investigate the mechanism of drug resistance. Salvicine (SAL) was used to reverse the drug resistance in a gemcitabine-resistant pancreatic cancer cell line (SW1990-GEM). RT-PCR, flow cytometry and MTT assay were employed to evaluate the effect of reversing drug resistance by salvicine. RESULTS: SW1990-GEM cells showed weak expression of P-glycoprotein (P-gp) revealed by immunohistochemistry, while its parental SW1990 cells were negative. Both cell lines did not express multidrug-resistance-related protein (MRP). As compared to the parental SW1990 cells, the mRNA expression of deoxycytidine kinase (dCK) was decreased while that of ribonucleotide reductase (RR) and mdr-1 was increased. With a concentration of 4 nmol/L, at one hr and 24 hr after SAL treatment, there was no change in mdr-1 mRNA expression, and the IC(50) of gemcitabine was 121.36 micromol/L and 121.64 micromol/L, respectively. However, when the concentration of SAL was increased to 30, 60, and 90 nmol/L, there was a dose-dependent down-regulation of mdr-1 mRNA expression in SW1990-GEM cells. The accumulation of rhodamine 123 was concomitantly increased, and the IC(50) of gemcitabine was correspondingly decreased. CONCLUSION: The resistance to gemcitabine of a pancreatic cancer cell line is due to decreased expression of dCK and increased expression of RR and mdr-1. Salvicine, only in toxic concentrations, can reverse the drug resistance by down-regulating mdr-1 gene and P-gp expression.