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

中国临床药理学与治疗学 ›› 2004, Vol. 9 ›› Issue (4): 403-410.

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

詹晓云¹, 任宇红^1,2, 张强¹, 卢艳花¹, 魏东芝¹, 刘建文¹

¹上海华东理工大学, 生物反应器国家重点实验室, 鲁华生物技术研究所, 上海200237;
²江西省南昌大学, 江西OAI 中德研究所, 南昌330047, 江西

收稿日期:2003-12-04 接受日期:2004-01-30 出版日期:2004-04-26 发布日期:2020-11-20

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

ZHAN Xiao-Yun¹, REN Yu-Hong^1,2, ZHANG Qiang¹, LU Yan-Hua¹, WEI Dong-Zhi¹, LIU Jian-Wen¹

¹StateKey Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China;
²Jiangxi-OAI Gemeinsames Institute, Nanchang University, Nanchang 330047, Jiangxi, China

Received:2003-12-04 Accepted:2004-01-30 Online:2004-04-26 Published:2020-11-20
Contact: WEI Dong-Zhi, male, doctor, professor, tutor of doctor,engaged in biotechnology.Tel:021-64252981 Fax:021-64250068 E-mail:dzhwei@ecust.edu.cn;LIU Jian-Wen, male, doctor, prof essor, tutor of doctor,engaged in study of screening of anti cancer drug and the eff iciency of active material.Tel:021-64253715 Fax:021-64250068 E-mail:liujian@ecust.edu.cn
About author:ZHAN Xiao-Yun, male, master in reading, engaged in tumor pharmacology.Tel:021-64253871 Fax:021-64250068 E-mail:zhanxy_ecust@citiz.net
Supported by:
Project supported by the National Natural Foundation (№30171088) and National Plan 863(№2001AA215261)

摘要/Abstract

摘要： 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。

关键词: 反义寡核苷酸, 偶联物, 细胞毒性, 多药抗性, P-gp 蛋白

Abstract: 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.

Key words: oligodeoxynucleotide, conjugate, cytotoxicity, multidrug resistance, P-glycoprotein

中图分类号:

詹晓云, 任宇红, 张强, 卢艳花, 魏东芝, 刘建文. 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[J]. 中国临床药理学与治疗学, 2004, 9(4): 403-410.

ZHAN Xiao-Yun, REN Yu-Hong, ZHANG Qiang, LU Yan-Hua, WEI Dong-Zhi, LIU Jian-Wen. A novel antisense oligodeoxynucleotide-doxorubicin conjugate against the multidrug resistance gene mdr1:inhibition to KB-A-1 cell and P-glycoprotein expression[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2004, 9(4): 403-410.

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