反义寡核苷酸-萘二酰亚胺偶联物对多药耐药性肿瘤细胞的抑制作用

目的：针对人表皮癌细胞阿霉素耐药株（KB－A－1）的多药耐药性问题，探讨天然反义寡核苷酸与寡核苷酸－萘二酰亚胺偶联物对多药耐工 肿瘤细胞的抑制作用及mdr1基因表达的影响，方法：利用S－烷基化反应成功地合成了反义寡核苷酸－萘二酰亚胺偶联物，并通过MTT法和ELISA法测定天然反义寡核苷酸与寡核苷酸－萘二酰亚胺偶联物对多药耐药物性肿瘤细胞的抑制作用。结果：作用靶点不同的反义寡核苷酸对KB－A－1细胞生长的抑制能力不同。针对mdr1基因翻译起始区的寡核苷酸片段1－4对KB－A－1细胞细胞生长的抑制率分别为13．34％、14．32％、26．00％和25．37％，其中片段3的效果最好（P＜0．01）。与单独使用反义寡核苷酸相比较，萘二酰亚胺对寡核苷酸的共价修饰 提高了反义寡核苷酸对KB－A－1细胞生长的抑制。凝胶电泳实验结果也表明萘二酰亚胺对寡核苷酸的化学修饰能提高寡核苷酸抵逆解的能力。寡核苷酸及寡核苷酸－萘二酰亚胺偶联物专一性地抑制KB－1－A细胞生长，而对非多药耐药性的KB－3－1细胞株无明显影响，是由于寡核苷酸及寡核苷酸－萘二酰亚胺偶联物选择性地抑制mdr1基因表达所致。ELISA法检测结果表明寡核苷酸寡核苷酸－萘二酰亚胺偶联物可使KB－A－1细胞膜表面的mdr1基因表达产物P-gp量降低，并且寡核苷酸－萘二酰亚胺偶联物P－gp表达的程度比天然片段强烈。结论：反义寡核苷酸可专一性地抑制mdr1基因表达，使多药耐性肿瘤细胞生长受到抑制。寡核苷酸与萘二酰亚胺共价偶联能提高天然反义寡核苷酸抑制多药耐性及抵抗核酸酶降解的能力。

Inhibition of Multidrug Resistance in Human Tumor Cell by Naphthyl Imide-Conjugated Antisense Oligonucleotide

Objective： This study was designed to investigate the effectiveness of natural and naphthyl imide-conjugated antisense oligonucleotide on inhibiting mdr1 gene expression and of overcome the problem of multidrug resistance in human epidemic carcinomata anti-adriamycin cells(KB-A-1). Methods： A novel naphthyl imide-conjugated mdr1 antisense oligonucleotide was synthesized by S-alkylation reaction. Compared with the natural one, the effectiveness of naphthyl imide-conjugated mdr1 antisense oligonucleotide on inhibiting the multidrug resistance was detected by MTT colometric assay and ELISA. Results： The abilities of antisense oligonucleotides to inhibit the cell growth of KB-A-1 mainly depended upon their targeted sequences selected. The inhibiting rates to KB-A-1 cells of antisense oligonucleotides (from Oligo Ⅰ to Oligo Ⅳ ) which bind the targeted sequences of the translation initiation site of mdr1 were 13.34％ ,14.32％ ,26 00％ ， and 25.37％ ， respectively. Among four oligonucleotides,the activity of Oligo Ⅲ to inhibit the growth of KB-A-1 cell was the highest (P<0.01). Compared with the unmodified one, the inhibition of antisense oligonucleotides to the growth of KB-A-1 cell was increased by conjugating with naphthyl imide. The ability of naphthyl imide-conjugated antisense oligonucleotide to resist serum-mediated nuclease was also increased as demonstrated by gel shift electrophoresis. The antisense oligonucleotide or its naphythyl imide conjugate could only inhibit the growth of KB-A-1 cells and had no effect on the growth of KB-3-1 cells. So it could be inferred that the inhibition of KB-A-1 cells was due to the repression of gene expression of mdr1 in KB-A-1 cells by antisense oligonucleotide or its naphthyl imide conjugate. ELISA showed that the P-glycoprotein expressions were more strongly inhibited by naphthyl imide-conjugated oligonucleotide than that by unmodified oligonucleotide. Conclusion: Inhibition of human tumor cell growth was due to the inhibition on mdr1 gene expression and the reversal of multidrug resistance by antisense oligonucleotide and its naphthyl imide conjugated. 1, 8-Naphthyl imide could improve the properties of natural oligonucleotide in inhibiting the multdrug resistance and resisting nuclease by covalently linkage at the end of oligonucleotide.