Molecular and Cellular Biochemistry

, Volume 455, Issue 1–2, pp 159–167 | Cite as

Concomitance of downregulated active caspase-3 and upregulated X-chromosome linked inhibitor of apoptosis protein as a sensitive diagnostic approach for breast cancer

  • Samir F. ZohnyEmail author
  • Mazin A. Zamzami
  • Mohamed El-Shinawi


We aimed to explore the efficacy of active caspase-3 and X-chromosome linked inhibitor of apoptosis protein (XIAP) as diagnostic markers for breast cancer. Furthermore, we examined the relationship between the examined parameters and clinicopathological factors. The current study involved 96 patients diagnosed with breast cancer and 40 patients had benign breast diseases. The expression of active caspase-3 was analyzed by both ELISA and Western blot, whereas the expression of XIAP was determined by ELISA in cell lysates. Active caspase-3 was significantly downregulated, while XIAP was markedly upregulated in patients with breast cancer in comparison to benign group. A significant negative correlation was observed between active caspase-3 and XIAP in breast cancer patients. Low active caspase-3 expression was associated with high grade, whereas, the high XIAP level was correlated with poorly differentiated tumors and late tumor stages. The sensitivity and specificity were 73.96% and 80.0% for active caspase-3, and, 70.83% and 82.5% for XIAP. A combination of active caspase-3 and XIAP provided a promising sensitivity of 88.54% and specificity of 90.0%. Our data indicate that active caspase-3 and XIAP could be substantial diagnostic markers for breast cancer patients.


Breast cancer Active caspase-3 XIAP Clinicopathological factors 



This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, under Grant No. G-48-130-38. The authors, therefore, acknowledge with thanks DSR for technical and financial support. The authors thank the Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt, for providing the histopathological data of the patients included in this study.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical standards

All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments.

Informed consent

Informed consent was obtained from all patients included in the study.


  1. 1.
    Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68:383–424Google Scholar
  2. 2.
    Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489Google Scholar
  3. 3.
    Atkinson EA, Barry M, Darmon AJ, Shostak I, Turner PC, Moyer RW, Bleackley RC (1998) Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. J Biol Chem 273:21261–21266Google Scholar
  4. 4.
    Stennicke HR, Jürgensmeier JM, Shin H, Deveraux Q, Wolf BB, Yang X, Zhou Q, Ellerby HM, Ellerby LM, Bredesen D, Green DR, Reed JC, Froelich CJ, Salvesen GS (1998) Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem 273:27084–27090Google Scholar
  5. 5.
    Han Z, Hendrickson EA, Bremner TA, Wyche JH (1997) A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. J Biol Chem 272:13432–13436Google Scholar
  6. 6.
    Devarajan E, Sahin AA, Chen JS, Krishnamurthy RR, Aggarwal N, Brun AM, Sapino A, Zhang F, Sharma D, Yang XH, Tora AD, Mehta K (2002) Down-regulation of caspase 3 in breast cancer: a possible mechanism for chemoresistance. Oncogene 21(57):8843–8851Google Scholar
  7. 7.
    Nassar A, Lawson D, Cotsonis G, Cohen C (2008) Survivin and caspase-3 expression in breast cancer: correlation with prognostic parameters, proliferation, angiogenesis, and outcome. Appl Immunohistochem Mol Morphol 16(2):113–120Google Scholar
  8. 8.
    Vakkala M, Pääkkö P, Soini Y (1999) Expression of caspases 3, 6 and 8 is increased in parallel with apoptosis and histological aggressiveness of the breast lesion. Br J Cancer 81(4):592–599Google Scholar
  9. 9.
    Nakopoulou L, Alexandrou P, Stefanaki K, Panayotopoulou E, Lazaris AC, Davaris PS (2001) Immunohistochemical expression of caspase-3 as an adverse indicator of the clinical outcome in human breast cancer. Pathobiology 69(5):266–273Google Scholar
  10. 10.
    O’Donovan N, Crown J, Stunell H, Hill AD, McDermott E, O’Higgins N, Duffy MJ (2003) Caspase 3 in breast cancer. Clin Cancer Res 9(2):738–742Google Scholar
  11. 11.
    Blázquez S, Sirvent JJ, Olona M, Aguilar C, Pelegri A, Garcia JF, Palacios J (2006) Caspase-3 and caspase-6 in ductal breast carcinoma: a descriptive study. Histol Histopathol 21(12):1321–1329Google Scholar
  12. 12.
    Grigoriev MY, Pozharissky KM, Hanson KP, Imyanitov EN, Zhivotovsky B (2002) Expression of caspase-3 and – 7 does not correlate with the extent of apoptosis in primary breast carcinomas. Cell Cycle 1(5):337–342Google Scholar
  13. 13.
    Deveraux QL, Reed JC (1999) IAP family proteins-suppressors of apoptosis. Genes Dev 13:239–252Google Scholar
  14. 14.
    Budhidarmo R, Day CL (2015) IAPs: Modular regulators of cell signalling. Semin Cell Dev Biol 39:80–90Google Scholar
  15. 15.
    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 24:645–655Google Scholar
  16. 16.
    Shiozaki EN, Chai J, Rigotti DJ, Riedl SJ, Li P, Srinivasula SM, Alnemri ES, Fairman R, Shi Y (2003) Mechanism of XIAP-mediated inhibition of caspase-9. Mol Cell 11:519–527Google Scholar
  17. 17.
    Suzuki Y, Nakabayashi Y, Takahashi R (2001) Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad Sci 98:8662–8667Google Scholar
  18. 18.
    Morizane Y, Honda R, Fukami K, Yasuda H (2005) X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO. J Biochem 137:125–132Google Scholar
  19. 19.
    Obexer P, Ausserlechner MJ (2014) X-linked inhibitor of apoptosis protein–a critical death resistance regulator and therapeutic target for personalized cancer therapy. Front Oncol 4:197Google Scholar
  20. 20.
    Jaffer S, Orta L, Sunkara S, Sabo E, Burstein DE (2007) Immunohistochemical detection of antiapoptotic protein X-linked inhibitor of apoptosis in mammary carcinoma. Hum Pathol 38(6):864–870Google Scholar
  21. 21.
    Zhang Y, Zhu J, Tang Y, Li F, Zhou H, Peng B, Zhou C, Fu R (2011) X-linked inhibitor of apoptosis positive nuclear labeling: a new independent prognostic biomarker of breast invasive ductal carcinoma. Diagn Pathol 6:49Google Scholar
  22. 22.
    Wang J, Liu Y, Ji R, Gu Q, Zhao X, Liu Y, Sun B (2010) Prognostic value of the X-linked inhibitor of apoptosis protein for invasive ductal breast cancer with triple-negative phenotype. Hum Pathol 41(8):1186–1195Google Scholar
  23. 23.
    Hinnis AR, Luckett JC, Walker RA (2007) Survivin is an independent predictor of short-term survival in poor prognostic breast cancer patients. Br J Cancer 96(4):639–645Google Scholar
  24. 24.
    Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A (eds) (2010) Breast. In: AJCC Cancer Staging Manual, 7th edn. Springer, New York, pp 345–377Google Scholar
  25. 25.
    Tavassoli FA, Devilee P (2003) World Health Organization classification of tumours. In: Pathology and genetics, tumours of the breast and female genital organs. IARC Press, Lyon, France, pp 19–23Google Scholar
  26. 26.
    Zohny SF, Baothman OA, El-Shinawi M, Al-Malki AL, Zamzami MA, Choudhry H (2017) The KIP/CIP family members p21Waf1/Cip1 and p57Kip2 as diagnostic markers for breast cancer. Cancer Biomark 18(4):413–423Google Scholar
  27. 27.
    Bradford NM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  28. 28.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 42–59Google Scholar
  29. 29.
    Evan GI, Vousden KH (2001) Proliferation, cell cycle and apoptosis in cancer. Nature 411(6835):342–348Google Scholar
  30. 30.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70Google Scholar
  31. 31.
    Chung C (2018) Restoring the switch for cancer cell death: targeting the apoptosis signaling pathway. Am J Health Syst Pharm 75(13):945–952Google Scholar
  32. 32.
    Moll UM, Zaika A (2001) Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett 493(2–3):65–69Google Scholar
  33. 33.
    Ding HF, Lin YL, McGill G, Juo P, Zhu H, Blenis J, Yuan J, Fisher DE (2000) Essential role for caspase-8 in transcription-independent apoptosis triggered by p53. J Biol Chem 275(49):38905–38911Google Scholar
  34. 34.
    Zhang Y, Wang Y, Gao W, Zhang R, Han X, Jia M, Guan W (2006) Transfer of siRNA against XIAP induces apoptosis and reduces tumor cells growth potential in human breast cancer in vitro and in vivo. Breast Cancer Res Treat 96(3):267–277Google Scholar
  35. 35.
    Yang L, Cao Z, Yan H, Wood WC (2003) Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis in human tumor cells: implication for cancer specific therapy. Cancer Res 63(20):6815–6824Google Scholar
  36. 36.
    Nestal de Moraes G, Delbue D, Silva KL, Robaina MC, Khongkow P, Gomes AR, Zona S, Crocamo S, Mencalha AL, Magalhães LM, Lam EW, Maia RC (2015) FOXM1 targets XIAP and survivin to modulate breast cancer survival and chemoresistance. Cell Signal 27(12):2496–2505Google Scholar
  37. 37.
    Yang X, Stennicke HR, Wang B, Green DR, Jänicke RU, Srinivasan A, Seth P, Salvesen GS, Froelich CJ (1998) Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and – 7. J Biol Chem 273(51):34278–34283Google Scholar
  38. 38.
    Yang XH, Edgerton S, Thor AD (2005) Reconstitution of caspase-3 sensitizes MCF-7 breast cancer cells to radiation therapy. Int J Oncol 26(6):1675–1680Google Scholar
  39. 39.
    McManus DC, Lefebvre CA, Cherton-Horvat G, St-Jean M, Kandimalla ER, Agrawal S, Morris SJ, Durkin JP, Lacasse EC (2004) Loss of XIAP protein expression by RNAi and antisense approaches sensitizes cancer cells to functionally diverse chemotherapeutics. Oncogene 23(49):8105–8117Google Scholar
  40. 40.
    Chaudhary AK, Yadav N, Bhat TA, O’Malley J, Kumar S, Chandra D (2016) A potential role of X-linked inhibitor of apoptosis protein in mitochondrial membrane permeabilization and its implication in cancer therapy. Drug Discov Today 21(1):38–47Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Biochemistry Department, Faculty of ScienceKing Abdulaziz UniversityJeddahKingdom of Saudi Arabia
  2. 2.Biochemistry Department, Faculty of ScienceAin Shams UniversityAbbassiaEgypt
  3. 3.General Surgery Department, Faculty of MedicineAin Shams UniversityAbbassiaEgypt

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