一种针对mdr1 基因的寡核苷酸-阿霉素偶联物:对KB-A-1 细胞的毒性及其抑制P-gp 蛋白表达的效果

目的:癌细胞膜上P-gp 糖蛋白的过量表达是肿瘤多药抗性的主要机制。人体内编码P-gp 糖蛋白的基因中仅有mdr1 涉及多药抗性。本研究中设计了针对mdr1 的反义核酸与阿霉素的偶联物, 并且对其细胞毒性进行了考察。同时对偶联物对人表皮癌细胞株KB-A-1 内的P-gp 蛋白的表达也做了分子水平上的研究。方法:使用MTT 法考察偶联物对KB-A-1 细胞的毒性。用HPLC 考察偶联物对细胞内阿霉素的积累量的影响。对于P-gp 蛋白表达的变化, 主要是通过RT-PCR 及WesternBlot 方法进行了研究。结果:偶联物的细胞毒性比寡核苷酸高。在低剂量的偶联物(0.5 μmol·L^-1) 的作用下, 细胞对阿霉素的敏感性提高。偶联物能有效的提高细胞内阿霉素的积累量。并且从RT-PCR 及WesternBlot看, 偶联物处理后的细胞内P-gp 表达最少。结论:选用合适的基团, 对反义核酸进行结构修饰能够较好的增强反义核酸的性能。采用阿霉素作为偶联基团尽管增强了细胞毒性, 但是在更大程度上增强了其抑制P-gp 蛋白的效力, 提高了肿瘤耐药性的逆转倍数, 具有一定的潜在应用价值。

A novel antisense oligodeoxynucleotide-doxorubicin conjugate against the multidrug resistance gene mdr1:inhibition to KB-A-1 cell and P-glycoprotein expression

AIM: Over-expression of P-glycoprotein (P-gp) on the surface of tumor cells that works as a membrane pump enhancing the drug efflux is regarded as a main candidate mechanism of multidrug resistance (MDR).And mdr1 is one of the two main genes that encode P-gp in human cells.In this study, a novel conjugate made of mdr1-anti-sense oligodeoxynucleotide (ODN) and a potent anticancer drug doxorubicin was constructed.The cytotoxicity of the conjugate as well as the effect of the conjugate on the modulation of P-gp-mediated MDR in KB-A-1 cell lines was studied.The molecular mechanism of its regulation effect was also investigated. METHODS: Multidrug resistant KB-A-1 cell lines were used.MTT was applied to measure the cytotoxicity of the conjugate and doxorubicin to KB-A-1 cells. Effect of the conjugate on intracellular doxorubicin accumulation was determined by HPLC.RT-PCR and Western blot were used to determine the expression of mdr1 gene and P-glycoprotein in KB-A-1 cells.RESULTS: The results showed that the conjugate exhibited more cytotoxicity than ODN, and also it could promote the cytotoxicity of doxorubicin to KB-A-1 cells.The IC50 of doxorubicin dropped from 24 to 18 μg·ml^-1.The results of RT-PCR and Western blot analysis illuminated that conjugate might down-regulate the P-gp expression.CONCLUSION: All the findings suggest that the conjugate is more potential for efficient reversal of MDR phenomenon than free ODN. The study indicated that such a conjugate should be a new feasible and efficient anti-sense reagent both in lab research and further in clinical therapy.