Cancer Chemotherapy and Pharmacology

, Volume 64, Issue 2, pp 307–316 | Cite as

Stable XIAP knockdown clones of HCT116 colon cancer cells are more sensitive to TRAIL, taxanes and irradiation in vitro

  • Kate Connolly
  • Richard Mitter
  • Morwenna Muir
  • Duncan Jodrell
  • Sylvie Guichard
Original Article



To develop a model of X-linked inhibitor of apoptosis (XIAP) down regulation in colorectal cancer cell lines. This may be used to determine whether combination strategies have clinical potential.


A series of clones were developed using short hairpin RNA (shRNA) against XIAP stably expressed in HCT116 cells. XIAP mRNA and protein levels were established by RT-PCR and Immunoblot, respectively. GeneChip microarrays confirmed XIAP knockdown and absence of compensation by other IAP members.


Four XIAP knockdown cell lines show 82–93% reduction in XIAP mRNA and 67–89% reduction in protein when compared to four luciferase control cell lines. XIAP knockdown sensitises cells to rhTRAIL by a factor of 3, to paclitaxel and docetaxel by a factor of >2 and, to a lesser extent, radiotherapy (20% enhancement).


Clinical trials with XIAP antisense continue, and these data suggest combination studies with agents such as rhTRAIL and taxanes should be undertaken.


XIAP HCT116 TRAIL Radiotherapy Paclitaxel Docetaxel Microarray 



AMP-activated protein kinase


Cellular inhibitor of apoptosis protein 1


Cellular inhibitor of apoptosis protein 2


Death receptor


Inhibitor of apoptosis protein


Non-small cell lung cancer


Neuronal apoptosis inhibitor protein


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Robust multichip average


RNA interference


Recombinant human TNF related apoptosis inducing ligand


Real time polymerase chain reaction


Short hairpin RNA


Sulforhodamine B


X-linked inhibitor of apoptosis protein



The authors would like to thank Michael Dodds and Susan Alexander for technical assistance, Aegera Therapeutics Inc for provision of the shRNA constructs and the Cancer Research UK GeneChip microarray service based in the Paterson Institute, University of Manchester for processing the microarray samples.

Conflict of interest statement

K Connolly was funded by Cancer Research UK (Grant number C96/A4743) and her salary was supported in part by Aegera Therapeutics Inc. The Edinburgh Cancer Research Centre received clinical trial support for the Phase I study AEG35156 (XIAP antisense) from Aegera Therapeutics Inc.


  1. 1.
    Debatin KM (2004) Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother 53(3):153–159PubMedCrossRefGoogle Scholar
  2. 2.
    Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388(6639):300–304PubMedCrossRefGoogle Scholar
  3. 3.
    Scott FL, Denault JB, Riedl SJ, Shin H, Renatus M, Salvesen GS (2005) XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs. EMBO J 24(3):645–655PubMedCrossRefGoogle Scholar
  4. 4.
    Shiozaki EN, Chai J, Rigotti DJ et al (2003) Mechanism of XIAP-mediated inhibition of caspase-9. Mol Cell 11(2):519–527PubMedCrossRefGoogle Scholar
  5. 5.
    Wright CW, Duckett CS (2005) Reawakening the cellular death program in neoplasia through the therapeutic blockade of IAP function. J Clin Invest 115(10):2673–2678PubMedCrossRefGoogle Scholar
  6. 6.
    Eckelman BP, Salvesen GS (2006) The human anti-apoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases. J Biol Chem 281(6):3254–3260PubMedCrossRefGoogle Scholar
  7. 7.
    Dohi T, Okada K, Xia F et al (2004) An IAP-IAP complex inhibits apoptosis. J Biol Chem 279(33):34087–34090PubMedCrossRefGoogle Scholar
  8. 8.
    Liu Z, Li H, Wu X et al (2006) Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells. Oncogene 25(59):7680–7690PubMedCrossRefGoogle Scholar
  9. 9.
    Schwab M, Reynders V, Loitsch S et al (2008) PPARgamma is involved in mesalazine-mediated induction of apoptosis and inhibition of cell growth in colon cancer cells. Carcinogenesis 29(7):1407–1414PubMedCrossRefGoogle Scholar
  10. 10.
    Tamm I, Kornblau SM, Segall H et al (2000) Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res 6(5):1796–1803PubMedGoogle Scholar
  11. 11.
    Krajewska M, Krajewski S, Banares S et al (2003) Elevated expression of inhibitor of apoptosis proteins in prostate cancer. Clin Cancer Res 9(13):4914–4925PubMedGoogle Scholar
  12. 12.
    Krajewska M, Kim H, Kim C et al (2005) Analysis of apoptosis protein expression in early-stage colorectal cancer suggests opportunities for new prognostic biomarkers. Clin Cancer Res 11(15):5451–5461PubMedCrossRefGoogle Scholar
  13. 13.
    Mizutani Y, Nakanishi H, Li YN et al (2007) Overexpression of XIAP expression in renal cell carcinoma predicts a worse prognosis. Int J Oncol 30(4):919–925PubMedGoogle Scholar
  14. 14.
    Liu SS, Tsang BK, Cheung AN et al (2001) Anti-apoptotic proteins, apoptotic and proliferative parameters and their prognostic significance in cervical carcinoma. Eur J Cancer 37(9):1104–1110PubMedCrossRefGoogle Scholar
  15. 15.
    Ferreira CG, van der Valk P, Span SW et al (2001) Expression of X-linked inhibitor of apoptosis as a novel prognostic marker in radically resected non-small cell lung cancer patients. Clin Cancer Res 7(8):2468–2474PubMedGoogle Scholar
  16. 16.
    Schimmer AD, Dalili S, Batey RA, Riedl SJ (2006) Targeting XIAP for the treatment of malignancy. Cell Death Differ 13(2):179–188PubMedCrossRefGoogle Scholar
  17. 17.
    Ranson M, Ward T, Cummings J et al (2005) A Phase 1 Trial of AEG35156 (XIAP antisense) administered as a 7-day continuous intravenous infusion in patients with advanced tumors. Clin Cancer Res 11(24, Part 2):8965s–9216sGoogle Scholar
  18. 18.
    McManus DC, Lefebvre CA, Cherton-Horvat G et al (2004) Loss of XIAP protein expression by RNAi and antisense approaches sensitizes cancer cells to functionally diverse chemotherapeutics. Oncogene 23(49):8105–8117PubMedCrossRefGoogle Scholar
  19. 19.
    Gentleman RC, Carey VJ, Bates DM et al (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5(10):R80PubMedCrossRefGoogle Scholar
  20. 20.
    R Development Core Team (2006) R: a language and environment for statistical computing. Available from:
  21. 21.
    Gautier L, Cope L, Bolstad BM, Irizarry RA (2004) affy–analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20(3):307–315PubMedCrossRefGoogle Scholar
  22. 22.
    Smyth G (2005) Limma: linear models for microarray data. Springer, New YorkGoogle Scholar
  23. 23.
    Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31(4):e15PubMedCrossRefGoogle Scholar
  24. 24.
    Smyth G (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology. 3, no 1:Article 3Google Scholar
  25. 25.
    Benjamini Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B(57):289–300Google Scholar
  26. 26.
    Skehan P, Storeng R, Scudiero D et al (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13):1107–1112PubMedCrossRefGoogle Scholar
  27. 27.
    Workman P, Twentyman P, Balkwill F, United Kingdom Co-Ordinating Committee on Cancer Research (UKCCCR) (1998) Guidelines for the welfare of animals in experimental neoplasia. Br J Cancer 77:1–10Google Scholar
  28. 28.
    Burstein E, Ganesh L, Dick RD et al (2004) A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J 23(1):244–254PubMedCrossRefGoogle Scholar
  29. 29.
    Takeuchi H, Kim J, Fujimoto A et al (2005) X-Linked inhibitor of apoptosis protein expression level in colorectal cancer is regulated by hepatocyte growth factor/C-met pathway via Akt signaling. Clin Cancer Res 11(21):7621–7628PubMedCrossRefGoogle Scholar
  30. 30.
    Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG (2004) A small molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death. Science 305(5689):1471–1474PubMedCrossRefGoogle Scholar
  31. 31.
    Liston P, Fong WG, Kelly NL et al (2001) Identification of XAF1 as an antagonist of XIAP anti-Caspase activity. Nat Cell Biol 3(2):128–133PubMedCrossRefGoogle Scholar
  32. 32.
    Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 8(3):613–621PubMedCrossRefGoogle Scholar
  33. 33.
    Hegde R, Srinivasula SM, Datta P et al (2003) The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein. J Biol Chem 278(40):38699–38706PubMedCrossRefGoogle Scholar
  34. 34.
    Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, Lipp J (1998) Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J Exp Med 188(1):211–216PubMedCrossRefGoogle Scholar
  35. 35.
    Harlin H, Reffey SB, Duckett CS, Lindsten T, Thompson CB (2001) Characterization of XIAP-deficient mice. Mol Cell Biol 21(10):3604–3608PubMedCrossRefGoogle Scholar
  36. 36.
    Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F (2004) X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells. Cancer Res 64(9):3006–3008PubMedCrossRefGoogle Scholar
  37. 37.
    Olayioye MA, Kaufmann H, Pakusch M, Vaux DL, Lindeman GJ, Visvader JE (2005) XIAP-deficiency leads to delayed lobuloalveolar development in the mammary gland. Cell Death Differ 12(1):87–90PubMedCrossRefGoogle Scholar
  38. 38.
    Schimmer AD (2004) Inhibitor of apoptosis proteins: translating basic knowledge into clinical practice. Cancer Res 64(20):7183–7190PubMedCrossRefGoogle Scholar
  39. 39.
    Motoshima H, Goldstein BJ, Igata M, Araki E (2006) AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol 574(Pt 1):63–71PubMedCrossRefGoogle Scholar
  40. 40.
    Marini P, Denzinger S, Schiller D et al (2006) Combined treatment of colorectal tumours with agonistic TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 and radiotherapy: enhanced effects in vitro and dose-dependent growth delay in vivo. Oncogene 25(37):5145–5154PubMedGoogle Scholar
  41. 41.
    Buchsbaum DJ, Zhou T, Grizzle WE et al (2003) Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model. Clin Cancer Res 9(10 Pt 1):3731–3741PubMedGoogle Scholar
  42. 42.
    Cao C, Mu Y, Hallahan DE, Lu B (2004) XIAP and survivin as therapeutic targets for radiation sensitization in preclinical models of lung cancer. Oncogene 23(42):7047–7052PubMedCrossRefGoogle Scholar
  43. 43.
    Vogler M, Durr K, Jovanovic M, Debatin KM, Fulda S (2007) Regulation of TRAIL-induced apoptosis by XIAP in pancreatic carcinoma cells. Oncogene 26(2):248–257PubMedCrossRefGoogle Scholar
  44. 44.
    Yang L, Cao Z, Yan H, Wood WC (2003) Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy. Cancer Res 63(20):6815–6824PubMedGoogle Scholar
  45. 45.
    Ravi R, Jain AJ, Schulick RD et al (2004) Elimination of hepatic metastases of colon cancer cells via p53-independent cross-talk between irinotecan and Apo2 ligand/TRAIL. Cancer Res 64(24):9105–9114PubMedCrossRefGoogle Scholar
  46. 46.
    Endo T, Abe S, Seidlar HB et al (2004) Expression of IAP family proteins in colon cancers from patients with different age groups. Cancer Immunol Immunother 53(9):770–776PubMedCrossRefGoogle Scholar
  47. 47.
    Kanzler S TT, Heinemann V et al (2005) Results of a phase 2 trial of HGS-ETR1 (agonistic human monoclonal antibody to TRAIL receptor 1) in subjects with relapsed or refractory colorectal cancer (CRC) [abstract 630]. Eur J Cancer Suppl 3:177Google Scholar
  48. 48.
    Plummer R, Attard G, Pacey S et al (2007) Phase 1 and pharmacokinetic study of lexatumumab in patients with advanced cancers. Clin Cancer Res 13(20):6187–6194PubMedCrossRefGoogle Scholar
  49. 49.
    LaCasse EC, Cherton-Horvat GG, Hewitt KE et al (2006) Preclinical characterization of AEG35156/GEM 640, a second-generation antisense oligonucleotide targeting X-linked inhibitor of apoptosis. Clin Cancer Res 12(17):5231–5241PubMedCrossRefGoogle Scholar
  50. 50.
    Park SJ, Wu CH, Gordon JD, Zhong X, Emami A, Safa AR (2004) Taxol induces caspase-10-dependent apoptosis. J Biol Chem 279(49):51057–51067PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Kate Connolly
    • 1
  • Richard Mitter
    • 2
  • Morwenna Muir
    • 1
  • Duncan Jodrell
    • 1
    • 3
  • Sylvie Guichard
    • 1
    • 4
  1. 1.University of Edinburgh Cancer Research CentreEdinburghUK
  2. 2.Bioinformatics and BiostatisticsCancer Research UKLondonUK
  3. 3.Department of Oncology, Addenbrooke’s HospitalUniversity of CambridgeCambridgeUK
  4. 4.AstraZenecaMacclesfieldUK

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