Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells

Cancer Gene Therapy volume 11, pages 309–316 (2004)Cite this article

Abstract

Antiapoptotic genes such as bcl-2 or xIAP may be responsible for resistance to apoptosis induced by cytotoxic drugs. The aim of this study was to investigate if downregulation of bcl-2 or xIAP by RNA interference (RNAi) would sensitize MCF-7 cells to etoposide and doxorubicin. FITC-siRNAs uptake was verified by fluorescence microscopy and downregulation of Bcl-2 or XIAP was confirmed by Western Blotting. Both siRNAs reduced the number of viable cells and increased cellular apoptosis. Treatment with siRNAs followed by treatment with etoposide or doxorubicin further reduced the number of viable cells, when compared to either of the treatments alone. Therefore, downregulation of bcl-2 or xIAP by RNAi enhances the effects of etoposide and doxorubicin.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Igney FH, Krammer PH . Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer. 2002;2:277–288.

    Article  CAS  PubMed  Google Scholar 

  2. Cory S, Adams JM . The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002;2:647–656.

    Article  CAS  PubMed  Google Scholar 

  3. Adams JM, Cory S . The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322–1326.

    Article  CAS  PubMed  Google Scholar 

  4. Salvesen GS, Duckett CS . IAP proteins: blocking the road to death's door. Nat Rev Mol Cell Biol. 2002;3:401–410.

    Article  CAS  PubMed  Google Scholar 

  5. Huang Z . Bcl-2 family proteins as targets for anticancer drug design. Oncogene. 2000;19:6627–6631.

    Article  CAS  PubMed  Google Scholar 

  6. Holcik M, Korneluk RG . XIAP, the guardian angel. Nat Rev Mol Cell Biol. 2001;2:550–556.

    Article  CAS  PubMed  Google Scholar 

  7. Verhagen AM, Coulson EJ, Vaux DL . Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs. Genome Biol. 2001;2:3009.1–3009.10.

    Article  Google Scholar 

  8. LaCasse EC, Baird S, Korneluk RG, et al. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene. 1998;17:3247–3259.

    Article  PubMed  Google Scholar 

  9. Wang J-L, Liu D, Zhang Z-J, et al. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci USA. 2000;97:7124–7129.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shangary S, Johnson DE . Recent advances in the development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family. Leukemia. 2003;17:1470–1481.

    Article  CAS  PubMed  Google Scholar 

  11. Pei XY, Dai Y, Grant S . The proteasome inhibitor bortezomib promotes mitochondrial injury and apoptosis induced by the small molecule Bcl-2 inhibitor HA14-1 in multiple myeloma cells. Leukemia. 2003;17:2036–2045.

    Article  CAS  PubMed  Google Scholar 

  12. Arnt CR, Chiorean MV, Heldebrant MP, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP1 in situ. J Biol Chem. 2002;277:44236–44243.

    Article  CAS  PubMed  Google Scholar 

  13. Waterhouse PM, Wang MB, Lough T . Gene silencing as an adaptative defence against viruses. Nature. 2001;411:834–842.

    Article  CAS  PubMed  Google Scholar 

  14. Sharp PA . RNA interference-2001. Genes Dev. 2001;15:485–490.

    Article  CAS  PubMed  Google Scholar 

  15. Tuschl T . RNA interference and small interfering RNAs. Chembiochemistry. 2001;2:239–245.

    Article  CAS  Google Scholar 

  16. Hammond SM, Caudy AA, Hannon GJ . Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet. 2001;2:110–119.

    Article  CAS  PubMed  Google Scholar 

  17. Bosher JM, Labouesse M . RNA interference: genetic wand and genetic watchdog. Nat Cell Biol. 2000;2:E31–E36.

    Article  CAS  PubMed  Google Scholar 

  18. Bernstein E, Denli AM, Hannon GJ . The rest is silence. RNA. 2001;7:1509–1521.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Dykxhoorn DM, Novina CD, Sharp PA . Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 2003;4:457–467.

    Article  CAS  PubMed  Google Scholar 

  20. Sullenger BA, Gilboa E . Emerging clinical applications of RNA. Nature. 2002;418:252–258.

    Article  CAS  PubMed  Google Scholar 

  21. Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with BCR/ABL fusion gene by RNA interference (RNAi). Oncogene. 2002;21:5716–5724.

    Article  CAS  PubMed  Google Scholar 

  22. Scherr M, Battmer K, Winkler T, et al. Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood. 2003;101:1566–1569.

    Article  CAS  PubMed  Google Scholar 

  23. Nieth C, Priebsch A, Stege A, et al. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi). FEBS Lett. 2003;545:144–150.

    Article  CAS  PubMed  Google Scholar 

  24. Cioca DP, Aoki Y, Kiyosawa K . RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther. 2003;10:125–133.

    Article  CAS  PubMed  Google Scholar 

  25. Abdelrahim M, Samudio I, Smith III R, et al. Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells. J Biol Chem. 2002;277:28815–28822.

    Article  CAS  PubMed  Google Scholar 

  26. Abdelrahim M, Smith R, Safe S . Aryl hydrocarbon receptor gene silencing with small inhibitory RNA differentially modulates Ah-responsiveness in MCF-7 and HepG2 cancer cells. Mol Pharmacol. 2003;63:1373–1381.

    Article  CAS  PubMed  Google Scholar 

  27. Srinivasula SM, Gupta S, Datta P, et al. Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2. J Biol Chem. 2003;278:31469–31472.

    Article  CAS  PubMed  Google Scholar 

  28. Friedrich K, Wieder T, Von Haefen C, et al. Overexpression of caspase-3 restores sensitivity for drug-induced apoptosis in breast cancer cell lines with acquired drug resistance. Oncogene. 2001;20:2749–2760.

    Article  CAS  PubMed  Google Scholar 

  29. Yang X-H, Sladek TL, Liu X, et al. Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis. Cancer Res. 2001;61:348–354.

    CAS  PubMed  Google Scholar 

  30. Vasconcelos MH, Beleza SS, Quirk C, et al. Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture. Cancer Lett. 2000;152:135–143.

    Article  CAS  PubMed  Google Scholar 

  31. Riedl SJ, Renatus M, Schwarzenbacher R, et al. Structural basis for the inhibition of caspase-3 by XIAP. Cell. 2001;104:791–800.

    Article  CAS  PubMed  Google Scholar 

  32. Srinivasula SM, Hegde R, Saleh A, et al. A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature. 2001;410:112–116.

    Article  CAS  PubMed  Google Scholar 

  33. Bilim V, Kasahara T, Hara N, et al. Role of XIAP in the malignant phenotype of transitional cell cancer (TCC) and the therapeutic activity of XIAP antisense oligonucleotides against multidrug-resistant TCC in vitro. Int J Cancer. 2003;103:29–37.

    Article  CAS  PubMed  Google Scholar 

  34. Sasaki H, Sheng Y, Kotsuji F, et al. Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells. Cancer Res. 2000;60:5659–5666.

    CAS  PubMed  Google Scholar 

  35. Harlin H, Reffey SB, Duckett CS, et al. Characterization of XIAP-deficient mice. Mol Cell Biol. 2001;21:3604–3608.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nakayama K, Nakayama K-I, Negishi I, et al. Targeted disruption of Bcl-2αβ in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia. Proc Natl Acad Sci USA. 1994;91:3700–3704.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Borner C . Diminished cell proliferation associated with the death-protective activity of Bcl-2. J Biol Chem. 1996;271:12695–12698.

    Article  CAS  PubMed  Google Scholar 

  38. Konopleva M, Tari AM, Estrov Z, et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins. Blood. 2000;95:3929–3938.

    CAS  PubMed  Google Scholar 

  39. Levkau B, Garton KJ, Ferri N, et al. xIAP induces cell-cycle arrest and activates nuclear factor-kB. Circul Res. 2001;88:282–290.

    Article  CAS  Google Scholar 

  40. de Almodovar CR, Ruiz-Ruiz C, Munoz-Pinedo C, et al. The differential sensitivity of Bcl-2-overexpressing human breast tumor cells to TRAIL or doxorubicin-induced apoptosis is dependent on Bcl-2 protein levels. Oncogene. 2001;20:7128–7133.

    Article  Google Scholar 

  41. Davis JM, Navolanic PM, Weinstein-Oppenheimer CR, et al. Raf-1 and bcl-2 induce distinct and common pathways that contribute to breast cancer drug resistance. Clin Cancer Res. 2003;9:1161–1170.

    CAS  PubMed  Google Scholar 

  42. Piché A, Grim J, Rancourt C, et al. Modulation of Bcl-2 protein levels by an intracellular anti-Bcl-2 single-chain antibody increases drug-induced cytotoxicity in the breast cancer cell line MCF-7. Cancer Res. 1998;58:2134–2140.

    PubMed  Google Scholar 

  43. Teixeira C, Reed JC, Pratt MAC . Estrogen promotes chemotherapeutic drug resistance by a mechanism involving Bcl-2 proto-oncogene expression in human breast cancer cells. Cancer Res. 1995;55:3902–3907.

    CAS  PubMed  Google Scholar 

  44. Del Bufalo D, Biroccio A, Trisciuoglio D, et al. Bcl-2 has differing effects on the sensitivity of breast cancer cells depending on the antineoplastic drug used. Eur J Cancer. 2002;38:2455–2462.

    Article  CAS  PubMed  Google Scholar 

  45. Holcik M, Yeh C, Korneluk RG, et al. Translational upregulation of X-linked inhibitor of apoptosis (XIAP) increases resistance to radiation induced cell death. Oncogene. 2000;19:4174–4177.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Catarina A Silva (MD) for technical support in the initial experiments. This work was supported by FCT (Portugal), project POCTI/FCB/42754/2001 — Programa Operacional “Ciência, Tecnologia, Inovação” do Quadro Comunitário de Apoio (QCA) III, including FEDER funding. Raquel T Lima is a recipient of a young researcher grant from the referred project.

Author information

Authors and Affiliations

  1. IPATIMUP — Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal

    Raquel T Lima, José E Guimarães, Clara Sambade & M Helena Vasconcelos

  2. Cancer Research UK, London, UK

    Luís M Martins

  3. Faculty of Medicine of the University of Porto and Hospital S João, Porto, Portugal

    José E Guimarães & Clara Sambade

  4. Department of Microbiology, Faculty of Pharmacy of the University of Porto, Porto, Portugal

    M Helena Vasconcelos

Authors
  1. Raquel T Lima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luís M Martins
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José E Guimarães
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Clara Sambade
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M Helena Vasconcelos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Helena Vasconcelos.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lima, R., Martins, L., Guimarães, J. et al. Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells. Cancer Gene Ther 11, 309–316 (2004). https://doi.org/10.1038/sj.cgt.7700706

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700706

Keywords

This article is cited by

Search

Advanced search

Quick links