反义寡核苷酸逆转卵巢癌细胞对拓扑替康耐药性的研究

Antisense Oligonucleotide Reverses Topotecan-Resistant Ovarian Cancer Cells

背景与目的:研究表明乳腺癌耐药蛋白(breastcancerresistanceprotein,BCRP)在卵巢癌拓扑替康(topotecan,TPT)耐药株A2780/TPT中存在高表达,它可能在卵巢癌耐药中起着重要的作用。本研究拟探讨BCRP反义寡核苷酸(antisenseoligonucleotide,ASODN)片段对A2780/TPT细胞耐药的逆转作用。方法:人工合成包含BCRPmRNA翻译起始位点的ASODN片段,并合成相应的正义寡核苷酸(senseoligonucleotide,SODN)片段作为对照。用脂质体Lipofect-2000将ASODN及SODN分别转染进A2780/TPT细胞,分别用RT-PCR法、流式细胞仪及MTT法检测A2780/TPT细胞经体外转染后,BCRPmRNA的表达、胞内罗丹明荧光强度及对TPT耐药指数的改变。结果:将ASODN/Lipofect-2000转染进A2780/TPT耐药细胞后,细胞内BCRPmRNA的表达较未转染细胞下降了59.42%(P<0.05),胞内罗丹明荧光强度由转染前的5.42增加到16.63(P<0.05),耐药指数由25降到5。而转染SODN/Lipofect-2000的A2780/TPT细胞中,上述指标无显著性变化。结论:转染ASODN/Lipofect-2000可部分逆转BCRP介导的卵巢癌细胞耐药。

BACKGROUND &OBJECTIVE:Breast cancer resistance protein (BCRP) w as overexpressed in topotecan (TPT)-selected human ovarian cancer cell line A2780 /TPT, strongly suggesting BCRP to be responsible for the drug-resistance of ova rian cancer. The current study was designed to investigate the reversal effect o f BCRP antisense oligonucleotide (ASODN) on topotecan-resistant A2780/TPT cells . METHODS: The antisense-phosphorothioate oligonu-cleotide including the trans lation initiation site of BCRP mRNA was artificially synthesized, and the sense oligonucleotide (SODN) corresponding to the ASODN was also synthesized as contro l. Lipofect-2000 (LF) was used for the transfer of either ASODN or SODN into A2 780/TPT cells. The changes of BCRP mRNA expression, intracellular fluorescence i ntensity of rhodamine and resistance index to topotecan of in vitro transfected A2780/TPT cells were detected respectively by reverse transcription-polymerase chain reaction (RT-PCR),flow cytometry (FCM),and methyl thiazolyl tetrazolium ( MTT) assay. RESULTS: The transfer of ASODN/LF into A2780/TPT cells resulted in:( 1)a 59.42%reduction of BCRP mRNA level (P< 0.05); (2)an obviously increased int racellular rhodamine fluorescence intensity from 5.42 to 16.63(P< 0.05); (3)a de creased resistance index to topotecan from 25 to 5 indicating sensitivity to top otecan in A2780/TPT cells recovered, as compared with non-transfected cell. But after transfecting SODN, no significant change could be measured. CONCLUSION: A SODN transfection may partly reverse BCRP-mediated drug-resistance of ovarian cancer cells.