Reversal MDR in Breast Carcinoma Cells by Transfection of Ribozyme Designed According the Secondary Structure of mdr1 mRNA

Peng Gao；Geng-Yin Zhou；Qing-Hui Zhang；Hong Li；Kun Mu；Yin-Ping Yuan；Jing Zhang；Bao-Heng Wang

multidrug resistance ； reversal ； ribozyme

The Chinese Journal of Physiology

49卷2期（2006 / 04 / 01）

96 - 103

英文

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The present study aims to investigate whether the ribozyme could reverse MDR in breast carcinoma cells. In this study, two GUC sites (GUC106 and GUC135) on the surface of mdr1 mRNA were selected according to the secondary structure of the 5'-region of mdr1 mRNA. The ribozyme gene RZ106 and RZ135 complementary to two sides bases of the target GUC were synthesized and cloned into the plasmid pEGFP-C1 which has EGFP (Enhanced Green Fluorescence Protein) as report gene and Kan/Neo as selection gene. After transfection with the recombinant plasmid and selected by G418, the stable cell clones were produced and used for detection. The alteration of mdr1 mRNA and P-gp in the treated cells was detected by RT-PCR, flow cytometry and Rh123 retention. The reversal efficiency of the drug resistance for adriamycin was determined by MTT assay. The results showed that after transfection with RZ106 and RZ135, the amount of the mdr1 mRNA and P-gp decreased significantly and the efflux function of P-gp was inhibited accordingly. Nine-fold and 16-fold reduction of resistance for adriamycin was observed in the two groups of treated cells. These results suggested that both ribozymes can reverse the MDR phenotype by inhibiting the expression of mdr1 mRNA and P-gp, and the RZ135 showed the better cleavage efficiency. The ribozyme strategy designed according the secondary structure of the target RNA could be a useful therapy for reversal of MDR.

1. Bevilacqua, P.C.(2004).Mechanism of catalytic RNA.Biopolymers,73,69-70.
2. Bratty, J.,Chartrand, P.,Ferbeyre, G.,Cedergren, R.(1993).The hammerhead RNA domain, a model ribozyme..Biochim. Biophys. Acta.,1216,345-359.
3. Cech, T.R.(2002).Ribozymes, the first 20 years..Biochem. Soc. Trans.,30,1162-1166.
4. Chalfie, M.(1995).Green fluorescent protein..Photochem. Photobiol.,62,651-656.
5. Chomczynski, P.,Sacchi, N.(1987).Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem.,162,156-159.
6. David, H. M.,Sabina, J.,Zuker, M.(1999).Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure..J. Mol. Biol.,288,911-940.
7. Fojo, T.,Bates, S.(2003).Strategies for reversing drug resistance.Oncogene,22,7512-7523.
8. Fruehauf, S.,Breems, D.A.,Knaan-shanzer, S.(1996).Frepuency analysis of multidrug resistency-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area froming cell assay and detection of vector-mediated p- glycoprotein expression by rhodamine123..Hum. Gene. Ther.,7,1219-1231.
9. Grude, P.,Conti, F.,Mennecier, D.,Louvel, A.,Houssin, D.,Weill, B.,Calmus, Y.(2002).MDR1 gene expression in hepatocellular carcinoma and the peritumoral liver of patients with and without cirrhosis.Cancer Lett.,186,107-113.
10. He, T.C.,Zhou, S.,Costa, L.T.,Yu, J.,Kinzler, K.W.,Vogelstein, B.(1998).A simplified system for generating recombinant adenoviruses..Proc. Natl. Acad. Sci. U.S.A.,95,2509-2514.
11. Ho, S.P.,Britton, D.H.,Stone, B.A.,Behrens, D.L.,Leffet, L.M.,Hobbs, F.W.,Miller, J.A.,Trainor, G.L.(1996).Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries.Nucleic Acids Res.,24,1901-1907.
12. Kaur, P.(2002).Multidrug resistance: Can different keys open the same lock?.Drug Resist. Updat.,5,61-64.
13. Kiehntopf, M.,Esquivel, E.L.,Brach, M.A.,Herrmann, F.(1995).Clinical applications of ribozymes..Lancet,345,1027-1031.
14. Kobayashi, H.,Takemura, Y.,Miyachi, H.(2001).Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics.Hum. Cell.,14,172-184.
15. Kobayashi, H.,Takemura, Y.,Ohnuma, T.(1992).Relationship between tumor cell density and drug concentration and the cytotoxic effects of doxorubicin or vincristine: mechanism of inoculum effects.Cancer Chemother. Pharmacol,31,6-10.
16. Kostenko, E.V.,Beabealashvilly, R.S.,Vlassov, V.V.,Zenkova, M.A.(2000).Secondary structure of the 5'-region of PGY1/MDR1 mRNA..FEBS Lett.,475,181-186.
17. Krishna, R.,Mayer, L.D.(2001).Modulation of P-glycoprotein (PGP) mediated multidrug resistance (MDR) using chemosensitizers: recent advances in the design of selective MDR modulators.Curr. Med. Chem. Anti-Canc. Agents,1,163-174.
18. Mahon, F.X.,Belloc, F.,Lagarde, V.,Chollet, C.,Moreau-Gaudry, F.,Reiffers, J.,Goldman, J.M.,Melo, J.V.(2003).MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models..Blood,101,2368-2373.
19. Marzolini, C.,Paus, E.,Buclin, T.,Kim, R.B.(2004).Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance.Clin. Pharmacol. Ther.,75,13-33.
20. Mcknight, K.L.,Heinz, B.A.(2003).RNA as a target for developing antivirals.Antivir. Chem. Chemother,14,61-73.
21. Pfrunder, A.,Gutmann, H.,Beglinger, C.,Drewe, J.(2003).Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1- MRP5) and hPXR in three different human colon carcinoma cell lines..J. Pharm. Pharmacol.,55,59-66.
22. Reichel, C.,Mathur, J.,Eckes, P.,Langenkemper, K.,Koncz, C.,Schell, J.,Reiss, B.,Maas, C.(1996).Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.Proc. Natl. Acad. Sci. U.S.A.,93,5888-5893.
23. Reinecke, P.,Schmitz, M.,Schneider, E.M.,Gabbert, H.E.,Gerharz, C.D.(2000).Multidrug resistance phenotype and paclitaxel (Taxol) sensitivity in human renal carcinoma cell lines of different histologic types..Cancer Invest,18,614-625.
24. Scott, W.G.,Klug, A.(1996).Ribozymes: structure and mechanism in RNA catalysis..Trends Biochem. Sci.,21,220-224.
25. Sioud, M.(2004).Ribozyme- and siRNA-mediated mRNA degradation: a general introduction.Methods Mol. Biol.,252,1-8.
26. Stewart, C.N.(2001).The utility of green fluorescent protein in transgenic plants..Plant Cell Rep.,20,376-382.
27. Turner, P.C.(2000).Ribozymes: Their design and use in cancer.Adv. Exp. Med. Biol,465,303-318.
28. Zampieri, L.,Bianchi, P.,Ruff, P.,Arbuthnot, P.(2002).Differential modulation by estradiol of P-glycoprotein drug resistance protein expression in cultured MCF7 and T47D breast cancer cells..Anticancer Res.,22,2253-2259.