Journal of Cancer Research and Clinical Oncology

, Volume 142, Issue 6, pp 1261–1271 | Cite as

Effects of paclitaxel on permanent head and neck squamous cell carcinoma cell lines and identification of anti-apoptotic caspase 9b

  • Regina MaushagenEmail author
  • Stefan Reers
  • Ann-Christin Pfannerstill
  • Angelina Hahlbrock
  • Roland Stauber
  • Ramtin Rahmanzadeh
  • Dirk Rades
  • Ralph Pries
  • Barbara Wollenberg
Original Article – Cancer Research



Paclitaxel is an effective chemotherapeutic agent against various human tumors inducing apoptosis via binding to β-tubulin of microtubules and arresting cells mainly in the G2/M phase of the cell cycle. However, the underlying specific molecular mechanisms of paclitaxel on head and neck squamous cell carcinoma (HNSCC) have not been identified yet.


The apoptotic effects and mechanisms of paclitaxel on different permanent HPV-negative HNSCC cell lines (UT-SCC-24A, UT-SCC-24B, UT-SCC-60A and UT-SCC-60B) were determined by flow cytometry assays, polymerase chain reaction analysis, immunofluorescence-based assays and sequencing studies.


Paclitaxel induced a G2/M arrest in HNSCC cell lines followed by an increased amount of apoptotic cells. Moreover, the activation of caspase 8, caspase 10 and caspase 3, and the loss of the mitochondrial outer membrane potential could be observed, whereas an activation of caspase 9 could barely be detected. The efficient activation of caspase 9 was not affected by altered methylation patterns. Our results can show that the promoter region of apoptotic protease activating factor 1 (Apaf-1) was not methylated in the HNSCC cell lines. By sequencing analysis two isoforms of caspase 9, the pro-apoptotic caspase 9 and the anti-apoptotic caspase 9b were identified. The anti-apoptotic caspase 9b is missing the catalytic site and acts as an endogenous inhibitor of apoptosis by blocking the binding of caspase 9 to Apaf-1 to form the apoptosome.


Our data indicate the presence of anti-apoptotic caspase 9b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.


Apoptosis Head and neck cancer Paclitaxel Caspases Caspase 9b 



This work was supported by Grants from the Werner and Klara Kreitz-Stiftung.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

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Supplementary material 1 (TIFF 2046 kb)
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Supplementary material 2 (TIFF 6262 kb)


  1. Adrain C, Slee EA, Harte MT, Martin SJ (1999) Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region. J Biol Chem 274:20855–20860CrossRefPubMedGoogle Scholar
  2. Ahmad ST, Arjumand W, Seth A, Saini AK, Sultana S (2012) Methylation of the APAF-1 and DAPK-1 promoter region correlates with progression of renal cell carcinoma in North Indian population. Tumour Biol 33:395–402. doi: 10.1007/s13277-011-0235-9 CrossRefPubMedGoogle Scholar
  3. Bala S et al (2000) Genetic analysis of the APAF1 gene in male germ cell tumors. Genes Chromosom Cancer 28:258–268CrossRefPubMedGoogle Scholar
  4. Baylin S, Bestor TH (2002) Altered methylation patterns in cancer cell genomes: cause or consequence? Cancer Cell 1:299–305CrossRefPubMedGoogle Scholar
  5. Chang YF, Li LL, Wu CW, Liu TY, Lui WY, P’Eng FK, Chi CW (1996) Paclitaxel-induced apoptosis in human gastric carcinoma cell lines. Cancer 77:14–18CrossRefPubMedGoogle Scholar
  6. Furukawa Y et al (2002) Apaf-1 is a mediator of E2F-1-induced apoptosis. J Biol Chem 277:39760–39768CrossRefPubMedGoogle Scholar
  7. Furukawa Y, Sutheesophon K, Wada T, Nishimura M, Saito Y, Ishii H, Furukawa Y (2005) Methylation silencing of the Apaf-1 gene in acute leukemia. Mol Cancer Res 3:325–334CrossRefPubMedGoogle Scholar
  8. Hakem R et al (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94:339–352CrossRefPubMedGoogle Scholar
  9. Hinz S, Kempkensteffen C, Weikert S, Schostak M, Schrader M, Miller K, Christoph F (2007) EZH2 polycomb transcriptional repressor expression correlates with methylation of the APAF-1 gene in superficial transitional cell carcinoma of the bladder. Tumour Biol 28:151–157. doi: 10.1159/000103380 CrossRefPubMedGoogle Scholar
  10. Hu Y, Benedict MA, Ding L, Nunez G (1999) Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. EMBO J 18:3586–3595. doi: 10.1093/emboj/18.13.3586 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Ibrado AM, Kim CN, Bhalla K (1998) Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells. Leukemia 12:1930–1936CrossRefPubMedGoogle Scholar
  12. Kuida K et al (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94:325–337CrossRefPubMedGoogle Scholar
  13. 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 91:479–489CrossRefPubMedGoogle Scholar
  14. Liebmann JE, Hahn SM, Cook JA, Lipschultz C, Mitchell JB, Kaufman DC (1993) Glutathione depletion by L-buthionine sulfoximine antagonizes taxol cytotoxicity. Cancer Res 53:2066–2070PubMedGoogle Scholar
  15. Lin HL, Chang YF, Liu TY, Wu CW, Chi CW (1998) Submicromolar paclitaxel induces apoptosis in human gastric cancer cells at early G1 phase. Anticancer Res 18:3443–3449PubMedGoogle Scholar
  16. Liu JR, Opipari AW, Tan L, Jiang Y, Zhang Y, Tang H, Nunez G (2002) Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance. Cancer Res 62:924–931PubMedGoogle Scholar
  17. Malladi S, Challa-Malladi M, Fearnhead HO, Bratton SB (2009) The Apaf-1*procaspase-9 apoptosome complex functions as a proteolytic-based molecular timer. EMBO J 28:1916–1925CrossRefPubMedPubMedCentralGoogle Scholar
  18. Manns J et al (2011) Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: activation of caspase-9 in the absence of Apaf-1. FASEB J 25:3250–3261. doi: 10.1096/fj.10-177527 CrossRefPubMedGoogle Scholar
  19. Mielgo A, Torres VA, Clair K, Barbero S, Stupack DG (2009) Paclitaxel promotes a caspase 8-mediated apoptosis through death effector domain association with microtubules. Oncogene 28:3551–3562CrossRefPubMedPubMedCentralGoogle Scholar
  20. Norbury CJ, Zhivotovsky B (2004) DNA damage-induced apoptosis. Oncogene 23:2797–2808CrossRefPubMedGoogle Scholar
  21. Park SJ, Wu CH, Gordon JD, Zhong X, Emami A, Safa AR (2004) Taxol induces caspase-10-dependent apoptosis. J Biol Chem 279:51057–51067. doi: 10.1074/jbc.M406543200 CrossRefPubMedGoogle Scholar
  22. Paska AV, Hudler P (2015) Aberrant methylation patterns in cancer: a clinical view. Biochem Med 25:161–176. doi: 10.11613/BM.2015.017 CrossRefGoogle Scholar
  23. Perkins CL, Fang G, Kim CN, Bhalla KN (2000) The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. Cancer Res 60:1645–1653PubMedGoogle Scholar
  24. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3):1458–1461. doi: 10.1038/nprot.2006.238 CrossRefPubMedGoogle Scholar
  25. Rowinsky EK, Donehower RC (1995) Paclitaxel (taxol). N Engl J Med 332:1004–1014CrossRefPubMedGoogle Scholar
  26. Schiff PB, Fant J, Horwitz SB (1979) Promotion of microtubule assembly in vitro by taxol. Nature 277:665–667CrossRefPubMedGoogle Scholar
  27. Seol DW, Billiar TR (1999) A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis. J Biol Chem 274:2072–2076CrossRefPubMedGoogle Scholar
  28. Shultz JC, Goehe RW, Wijesinghe DS, Murudkar C, Hawkins AJ, Shay JW, Minna JD, Chalfant CE (2010) Alternative splicing of caspase 9 is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation of SRp30a. Cancer Res 70(2):9185–9196. doi: 10.1158/0008-5472.CAN-10-1545 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Soengas MS et al (2001) Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 409:207–211. doi: 10.1038/35051606 CrossRefPubMedGoogle Scholar
  30. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1:949–957CrossRefPubMedGoogle Scholar
  31. Srinivasula SM, Ahmad M, Guo Y, Zhan Y, Lazebnik Y, Fernandes-Alnemri T, Alnemri ES (1999) Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res 59:999–1002PubMedGoogle Scholar
  32. Stepczynska A, Lauber K, Engels IH, Janssen O, Kabelitz D, Wesselborg S, Schulze-Osthoff K (2001) Staurosporine and conventional anticancer drugs induce overlapping, yet distinct pathways of apoptosis and caspase activation. Oncogene 20:1193–1202. doi: 10.1038/sj.onc.1204221 CrossRefPubMedGoogle Scholar
  33. Sugimura M, Sagae S, Ishioka S, Nishioka Y, Tsukada K, Kudo R (2004) Mechanisms of paclitaxel-induced apoptosis in an ovarian cancer cell line and its paclitaxel-resistant clone. Oncology 66:53–61. doi: 10.1159/000076335 CrossRefPubMedGoogle Scholar
  34. Vegran F, Boidot R, Solary E, Lizard-Nacol S (2011) A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly. PLoS ONE 6:e29058. doi: 10.1371/journal.pone.0029058 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Vermorken JB (2005) Medical treatment in head and neck cancer. Ann Oncol 16(Suppl. 2):ii258–ii264. doi: 10.1093/annonc/mdi735 PubMedGoogle Scholar
  36. Wang TH, Wang HS, Soong YK (2000) Paclitaxel-induced cell death: where the cell cycle and apoptosis come together. Cancer 88:2619–2628CrossRefPubMedGoogle Scholar
  37. Watanabe T et al (2003) Frequent LOH at chromosome 12q22-23 and Apaf-1 inactivation in glioblastoma. Brain Pathol 13:431–439CrossRefPubMedGoogle Scholar
  38. Yamamoto H, Gil J, Schwartz S Jr, Perucho M (2000) Frameshift mutations in Fas, Apaf-1, and Bcl-10 in gastro-intestinal cancer of the microsatellite mutator phenotype. Cell Death Differ 7:238–239CrossRefPubMedGoogle Scholar
  39. Yu J et al (2002) Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC Cancer 2:29CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Regina Maushagen
    • 1
    Email author
  • Stefan Reers
    • 2
  • Ann-Christin Pfannerstill
    • 3
  • Angelina Hahlbrock
    • 4
  • Roland Stauber
    • 4
  • Ramtin Rahmanzadeh
    • 5
  • Dirk Rades
    • 6
  • Ralph Pries
    • 1
  • Barbara Wollenberg
    • 1
  1. 1.Department of Otorhinolaryngology, Clinic for ENT and HNSUniversity Hospital of Schleswig-HolsteinLübeckGermany
  2. 2.Department of Cardiology and AngiologyUniversity Hospital MünsterMünsterGermany
  3. 3.Department for Nephrology and HypertensionUniversity Hospital of Schleswig-HolsteinKielGermany
  4. 4.Department of Molecular OncologyUniversity Medical Center MainzMainzGermany
  5. 5.Institute of Biomedical OpticsUniversity of LübeckLübeckGermany
  6. 6.Department of Radiation OncologyUniversity Hospital of Schleswig-HolsteinLübeckGermany

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