卵巢癌细胞拓扑替康耐药机制的探讨

目的　探讨人卵巢癌细胞对拓扑替康 (TPT)的耐药机制。方法　流式细胞仪检测卵巢癌TPT耐药细胞与亲本细胞的胞内罗丹明 (Rh12 3)荧光强度 ,RT PCR法检测各膜转运蛋白 (P gp、MRP、BCRP)的基因表达。将包含BCRPmRNA翻译起始位点的反义寡核苷酸 (ASODN)片段转染进耐药细胞 ,分别检测耐药细胞经体外转染后 ,BCRP的基因表达及胞内Rh12 3荧光强度的改变。结果　耐药株的胞内Rh12 3荧光强度是亲本细胞的 31.19% (P <0 .0 1)。耐药株中无P 糖蛋白 (P gp)的基因表达 ;多药耐药相关蛋白 (MRP)基因有极微弱表达 ,相对表达值为 0 .0 5 7;而BCRP基因在耐药株中高表达 ,相对表达值为 0 .6 6 ,亲本细胞不表达BCRP基因。将ASODN转染进耐药细胞后 ,BCRP的基因表达显著下降了 5 9.4 2 % (P <0 .0 5 ) ,胞内Rh12 3荧光强度由 5 .4 2增加到 16 .6 3(P <0 .0 5 )。结论　BCRP的高表达致胞内化疗药物浓度减少 ,是卵巢癌细胞对TPT耐药的主要原因。

The mechanism of topotecan resistance in ovarian cancer cell line

ObjectiveTo study the mechanism of topotecan (TPT) resistance in ovarian cancer cell line. MethodsA TPT-resistant ovarian cancer cell line A2780/TPT established in this laboratory was used in this study. Intracellular rhodamine fluorescence intensity of the TPT-resistant cells and parental cells were measured by flow cytometry. The gene expression of membrance protein transporter such as transporter P-glycoprotein (P-gp), multidrug resistance associated protein (MRP), breast cancer resistance protein(BCRP) was evaluated by RT-PCR. The antisense-phosphorothioate oligonucleotide (ASODN) including a translation initiation site of BCRP mRNA was transfered into resistant cells by liposome. ResultsIntracellular rhodamine fluorescence intensity of the resistant cells was 31.19% of that in the parental cells ( P <0.01). No expression of P-gp was demonstrated, and that of MRP was very weak in the TPT-resistant cells(relative expression value=0.057). BCRP was overexpressed in the TPT-resistant cells (relative expression=0.66), but not in the parental cells. Transfer of ASODN into resistant cells resulted in a 59.42% reduction of BCRP gene expression ( P <0.05) and an obviously increased intracellular rhodamine fluorescence intensity from 5.42 to 16.63 ( P <0.05). ConclusionThe overexpression of BCRP which mediated drug efflux may play an important role in the induction of TPT-resistance in ovarian cancer.