Pharmaceutical Research

, Volume 28, Issue 10, pp 2516–2529 | Cite as

siRNA-Mediated Down-Regulation of P-glycoprotein in a Xenograft Tumor Model in NOD-SCID Mice

  • Meysam Abbasi
  • Hamidreza Montazeri Aliabadi
  • Elaine H. Moase
  • Afsaneh Lavasanifar
  • Kamaljit Kaur
  • Raymond Lai
  • Charles Doillon
  • Hasan Uludağ
Research Paper



The efficacy of chemotherapy is decreased due to over-expression of the drug transporter P-glycoprotein (P-gp). This study was conducted to determine the feasibility of down-regulating tumor P-gp levels with non-viral siRNA delivery in order to sensitize the tumors to drug therapy.


P-gp over-expressing MDA435/LCC6 MDR1 cells were used to establish xenografts in NOD-SCID mouse. Cationic polymers polyethylenimine (PEI) and stearic acid-substituted poly-L-lysine (PLL-StA) were formulated with P-gp- specific siRNAs and delivered intratumorally to explore the feasibility of P-gp down-regulation in tumors. Intravenous Doxil™ was administered to investigate tumor growth.


PEI and PLL-StA effectively delivered siRNA to MDA435/LCC6 MDR1 cells in vitro to reduce P-gp expression for 3 days. Intratumoral injection of siRNA with the carriers resulted in 60-80% and 20–32% of siRNA retention in tumors after 24 and 96 hr, respectively. This led to ~29.0% and ~61.5% P-gp down-regulation with PEI- and PLL-StA-mediated siRNA delivery, respectively. The P-gp down-regulation by intratumoral siRNA injection led to better response to systemic Doxil™ treatment, resulting in slowed tumor growth in originally doxorubicin-resistant tumors.


Effective P-gp down-regulation was feasible with polymeric siRNA delivery in a xenograft model, resulting in an enhanced response to the drug therapy.


multidrug resistance non-viral siRNA delivery P-glycoprotein polymeric biomaterials xenograft 





Hank’s Balanced Salt Solution


multi-drug resistance


multi-drug resistance gene 1 expressing cells


non-obese/severe combined immunodeficient






stearic acid substituted poly-L-lysine


short interfering RNA


wild-type cells



Financial support for this project was provided by the Natural Sciences and Engineering Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR). Equipment support was provided by the Alberta Heritage Foundation for Medical Research (AHFMR) and Alberta Advanced Education & Technology. We thank Ms. Vanessa Incani for preparing the lipid-substituted PLL-StA, and Dr. Richard Clarke (Georgetown University, DC, USA) for the cell line used for this study.

Supplementary material

11095_2011_480_MOESM1_ESM.doc (52 kb)
Supplemetary Material (DOC 51 kb)


  1. 1.
    Kerbel RS. Molecular and physiologic mechanisms of drug resistance in cancer: an overview. Canc Metastasis Rev. 2001;20:1–2.CrossRefGoogle Scholar
  2. 2.
    Linardi RL, Natalini CC. Multi-drug resistance (MDR1) gene and P-glycoprotein influence on pharmacokinetic and pharmacodynamic of therapeutic drugs. Ciência Rural. 2006;36:336–41.CrossRefGoogle Scholar
  3. 3.
    Eckford PDW, Sharom FJ. ABC efflux pump-based resistance to chemotherapy drugs. Chem Rev. 2009;109:2989–3011.PubMedCrossRefGoogle Scholar
  4. 4.
    Higgins CF, Gottesman MM. Is the multidrug transporter a flippase? Trends Biochem Sci. 1992;17:18–21.PubMedCrossRefGoogle Scholar
  5. 5.
    Loo TW, Clarke DM. Recent progress in understanding the mechanism of P-glycoprotein mediated drug efflux. J Membr Biol. 2005;206:173–85.PubMedCrossRefGoogle Scholar
  6. 6.
    Schinkel AH, Borst P. Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P-glycoprotein. Int J Cancer. 1993;55:478–84.PubMedCrossRefGoogle Scholar
  7. 7.
    Chen Y, Simon SM. In situ biochemical demonstration that P-glycoprotein acts a drug efflux pump with broad specificity. J Cell Biol. 2000;148:5863–70.Google Scholar
  8. 8.
    Beaulieu E, Demeule M, Ghitescu L, Beliveau R. P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. Biochem J. 1997;326:539–44.PubMedGoogle Scholar
  9. 9.
    Van Zuylen L, Nooter K, Sparreboom A, Verweij J. Development of multidrug-resistance converters: sense or nonsense? Investig New Drugs. 2000;18:205–20.CrossRefGoogle Scholar
  10. 10.
    Thomas H, Coley HM. Overcoming multidrug resistance in cancer: P-gp modulators. Cancer Cont. 2003;10:159–65.Google Scholar
  11. 11.
    Kim SH, Jeong JH, Lee SH, Kim SW, Park TG. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. J Contr Release. 2008;129:107–16.CrossRefGoogle Scholar
  12. 12.
    Stierlé V, Laigle A, Jollés B. Modulation of MDR1 gene expression in multidrug resistant MCF7 cells by low concentrations of small interfering RNAs. Biochem Pharmacol. 2005;70:1424–30.PubMedGoogle Scholar
  13. 13.
    Nieth C, Priebsch A, Stege A, Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi). FEBS Lett. 2003;545:144–50.PubMedCrossRefGoogle Scholar
  14. 14.
    Akhtar S, Benter IF. Nonviral delivery of synthetic siRNAs in vivo. J Clin Invest. 2007;117:3623–32.PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang T, Guan M, Jin HY, Lu Y. Reversal of multidrug resistance by small interfering double-stranded RNAs in ovarian cancer cells. Gynecol Oncol. 2005;97:501–7.PubMedCrossRefGoogle Scholar
  16. 16.
    Xing H, Wang S, Weng D, Chen G, Yang X, Zhou J, et al. Knock-down of P-glycoprotein reverses taxol resistance in ovarian cancer multicellular spheroids. Oncol Rep. 2007;17:117–22.PubMedGoogle Scholar
  17. 17.
    Xu D, McCarty D, Fernandes A, Fisher M, Samulski RJ, Juliano RL. Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector. Mol Ther. 2005;11:523–30.PubMedCrossRefGoogle Scholar
  18. 18.
    Huaa J, Mutcha DJ, Herzog TJ. Stable suppression of MDR-1 gene using siRNA expression vector to reverse drug resistance in a human uterine sarcoma cell line. Gynecol Oncol. 2005;98:31–8.CrossRefGoogle Scholar
  19. 19.
    Yague E, Higgins CF, Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1. Gene Ther. 2004;11:1170–4.PubMedCrossRefGoogle Scholar
  20. 20.
    Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol. 2003;21:635–7.PubMedCrossRefGoogle Scholar
  21. 21.
    Wang Y, Hu JK, Krol A, Li YP, Li CY, Yuan F. Systemic dissemination of viral vectors during intratumoral injection. Mol Cancer Ther. 2003;2:1233–42.PubMedGoogle Scholar
  22. 22.
    Buyens K, Lucas B, Raemdonck K, Braeckmans K, Vercammen J, Hendrix J, et al. A fast and sensitive method for measuring the integrity of siRNA-carrier complexes in full human serum. J Contr Release. 2008;18:67–76.CrossRefGoogle Scholar
  23. 23.
    Song YK, Liu F, Chu S, Liu D. Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum Gene Ther. 1997;8:1585–94.PubMedCrossRefGoogle Scholar
  24. 24.
    Ruiz FE, Clancy JP, Perricone MA, Perricone MA, Bebok Z, Hong JS, et al. A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis. Hum Gene Ther. 2001;12:751–61.PubMedCrossRefGoogle Scholar
  25. 25.
    Scheule RK, George JA, Bagley RG, Marshall J, Kaplan JM, Akita GY, et al. Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung. Hum Gene Ther. 1997;8:689–707.PubMedCrossRefGoogle Scholar
  26. 26.
    Abbasi M, Lavasanifar A, Berthiaume LC, Weinfeld M, Uludağ H. Cationic polymer mediated siRNA delivery for P-glycoprotein (P-gp) down-regulation in tumor cells. Cancer. 2010;116:5544–54.PubMedCrossRefGoogle Scholar
  27. 27.
    Liu C, Zhao G, Liu J, Ma N, Chivukula P, Perelman L, et al. Novel biodegradable lipid nano complex for siRNA delivery significantly improving the chemosensitivity of human colon cancer stem cells to paclitaxel. J Contr Release. 2009;140:277–83.CrossRefGoogle Scholar
  28. 28.
    Alshamsan A, Haddadi A, Incani V, Samuel J, Lavasanifar A, Uludağ H. Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethyleneimine. Mol Pharma. 2009;6:121–33.CrossRefGoogle Scholar
  29. 29.
    Abbasi M, Uludağ H, Incani V, Hsu CYM, Jeffery A. Further investigation of lipid-substituted Poly(L-Lysine) polymers for transfection of human skin fibroblasts. Biomacromolecules. 2008;9:1618–30.PubMedCrossRefGoogle Scholar
  30. 30.
    Rae JM, Creighton CJ, Meck JM, Haddad BR, Johnson MD. MDA-MB-435 cells are derived from M14 melanoma cells-a loss for breast cancer, but a boon for melanoma research. Breast Cancer Res Treat. 2007;104:13–9.PubMedCrossRefGoogle Scholar
  31. 31.
    Shen H, Pan Y. Reversal of multidrug resistance of gastric cancer cells by downregulation of TSG101 with TSG101siRNA. Cancer Biol Ther. 2004;3:561–5.PubMedGoogle Scholar
  32. 32.
    Theyer G, Schirmböck M, Thalhammer T, Sherwood ER, Baumgartner G, Hamilton G. Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines. J Urol. 1993;150:1544–7.PubMedGoogle Scholar
  33. 33.
    Persengiev SP, Zhu X, Green MR. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA. 2004;10:12–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Semizarov D, Frost L, Sarthy A, Kroeger P, Halbert DN, Fesik SW. Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA. 2003;100:6347–52.PubMedCrossRefGoogle Scholar
  35. 35.
    Boese Q, Leake D, Reynolds A, Read S, Scaringe SA, Marshall WS, et al. Mechanistic insights aid computational short interfering RNA design. Meth Enzymol. 2005;392:73–96.PubMedCrossRefGoogle Scholar
  36. 36.
    Ji J, Wernli M, Klimkait T, Erb P. Enhanced gene silencing by the application of multiple specific small interfering RNAs. FEBS Lett. 2003;552:247–52.PubMedCrossRefGoogle Scholar
  37. 37.
    Holen T, Amarzguioui M, Wiiger MT, Babaie E, Prydz H. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 2002;30:1757–66.PubMedCrossRefGoogle Scholar
  38. 38.
    Shi Z, Liang Y, Chen Z, Wang X, Wang X, Ding Y, et al. Reversal of MDR1/P-glycoprotein-mediated multidrug resistance by vector-based RNA interference in vitro and in vivo. Canc Biol Ther. 2006;5:39–47.CrossRefGoogle Scholar
  39. 39.
    Jiang Z, Zhao P, Zhou Z, Liu J, Qin L, Wang H. Using attenuated Salmonella Typhi as tumor targeting vector for MDR1 siRNA delivery. Canc Biol Ther. 2007;6:555–60.CrossRefGoogle Scholar
  40. 40.
    Xiao H, Wu Z, Shen H, Luo A-L, Yang Y-F, Li X-B, et al. In vivo reversal of P-glycoprotein-mediated multidrug resistance by efficient delivery of Stealth™ RNAi. Basic Clin Pharmacol Toxicol. 2008;103:342–8.PubMedCrossRefGoogle Scholar
  41. 41.
    Patil YB, Swaminathan SK, Sadhukha T, Ma L, Panyam J. The use of nanoparticle-mediated targeted gene silencing and drug delivery to overcome tumor drug resistance. Biomaterials. 2010;31:358–65.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Meysam Abbasi
    • 1
  • Hamidreza Montazeri Aliabadi
    • 2
  • Elaine H. Moase
    • 3
  • Afsaneh Lavasanifar
    • 2
    • 3
  • Kamaljit Kaur
    • 3
  • Raymond Lai
    • 4
  • Charles Doillon
    • 5
  • Hasan Uludağ
    • 1
    • 2
    • 3
  1. 1.Department of Biomedical Engineering, Faculty of MedicineUniversity of AlbertaEdmontonCanada
  2. 2.Department of Chemical & Materials Engineering Faculty of Engineering,University of AlbertaEdmontonCanada
  3. 3.Faculty of Pharmacy and Pharmaceutical SciencesUniversity of AlbertaEdmontonCanada
  4. 4.Department of Laboratory Medicine & Pathology, Faculty of MedicineUniversity of AlbertaEdmontonCanada
  5. 5.CHUL’s Research Centre, CHUQLaval UniversitySilleryCanada

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