Regina Maushagen1, Stefan Reers2, Ann-Christin Pfannerstill3, Angelina Hahlbrock4, Roland Stauber4, Ramtin Rahmanzadeh5, Dirk Rades6, Ralph Pries7, Barbara Wollenberg7. 1. Department of Otorhinolaryngology, Clinic for ENT and HNS, University Hospital of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany. regina.maushagen@gmail.com. 2. Department of Cardiology and Angiology, University Hospital Münster, Albert-Schweitzer-Campus 1, 48149, Münster, Germany. 3. Department for Nephrology and Hypertension, University Hospital of Schleswig-Holstein, Campus Kiel, Schittenhelmstrasse 12, 24105, Kiel, Germany. 4. Department of Molecular Oncology, University Medical Center Mainz, Langenbeckstrasse 1, 55131, Mainz, Germany. 5. Institute of Biomedical Optics, University of Lübeck, Peter-Monnik-Weg 4, 23562, Lübeck, Germany. 6. Department of Radiation Oncology, University Hospital of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany. 7. Department of Otorhinolaryngology, Clinic for ENT and HNS, University Hospital of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany.
Abstract
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our data indicate the presence of anti-apoptotic caspase 9b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.
PURPOSE:Paclitaxel is an effective chemotherapeutic agent against various humantumors 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. METHODS: 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. RESULTS: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. CONCLUSION: Our data indicate the presence of anti-apoptotic caspase 9b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.
Entities:
Keywords:
Apoptosis; Caspase 9b; Caspases; Head and neck cancer; Paclitaxel
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