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A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53mut exhibits ATP-binding cassette transporter upregulation and high glutathione levels

  • Original Article – Cancer Research
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Abstract

Purpose

Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53mut_c) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53mut_n). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53mut.

Methods

Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53mut_c cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels.

Results

Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53mut_c HNSCC cells. Furthermore, p53mut_c cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53mut_c cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells.

Conclusions

The observations in this study point to a major role of p53mut_c in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.

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Acknowledgments

Technical assistance by Ms. Roswitha Peldszus and Ms. Maria Sadowski (Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Marburg, Germany) was greatly appreciated. The authors thank Dr. Florian Finkernagel (Institute for Molecular Biology and Tumor Research, Philipps University, Marburg, Germany) and Mr. Tomi Bähr-Ivacevic (Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany) for their help during microarray analysis. The authors thank Dr. Michael Pütz (Department of Neurology, University Hospital Giessen and Marburg, Campus Marburg, Marburg, Germany) for the use of their real-time thermal PCR cycler. R.M. was supported by a research grant of the University Medical Center Giessen and Marburg. C. B was supported by a grant of the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe 210).

Conflict of interest

All authors have no conflict of interest.

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Authors and Affiliations

  1. Department of Otolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Baldingerstrasse, 35033, Marburg, Germany

    Manuel Tonigold, Annette Rossmann, Marie Meinold, Chengzhong Cai, Fiona R. Rodepeter, Jochen A. Werner & Robert Mandic

  2. Institute of Anatomy and Cell Biology, Philipps University, Robert-Koch-Strasse 8, 35037, Marburg, Germany

    Annette Rossmann & Michael Bette

  3. Department of Hematology, Oncology and Immunology, University Hospital Giessen and Marburg, Campus Marburg, Baldingerstrasse, 35033, Marburg, Germany

    Melanie Märken, Katharina Henkenius, Gavin Giel, Andreas Neubauer & Cornelia Brendel

  4. Department of Molecular Oncology, Philipps-University, Hans-Meerwein-Str. 3, 35043, Marburg, Germany

    Anne C. Bretz & Thorsten Stiewe

  5. Genomics Core Facility, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117, Heidelberg, Germany

    Vladimir Beneš

  6. Department of Otorhinolaryngology - Head and Neck Surgery, Turku University and Turku University Central Hospital, P.O.Box 52, 20521, Turku, Finland

    Reidar Grénman

  7. Department of Otolaryngology-HNS, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI, 48109, USA

    Thomas E. Carey

  8. Institute of Pathology, Charité - Universitätsmedizin Berlin, Campus Mitte, Charitéplatz 1, 10117, Berlin, Germany

    Hermann Lage

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  1. Manuel Tonigold
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Correspondence to Robert Mandic.

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Manuel Tonigold and Annette Rossmann have contributed equally to this work.

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432_2014_1727_MOESM1_ESM.tif

Supplementary material Western blot analysis of whole cell lysates derived from UM-SCC-3p53mut_n and UT-SCC-26Ap53mut_c cells, treated for 2 days with 3.125, 6.25 or 12.5 µM CDDP in the presence or absence of 100 µM MK571, was tested for expression of MAP LC3β, p21CIP1/Waf1, caspase-3, p53 and β-actin. Bands derived from three independent Western blots were quantified and normalized to β-actin as described in the Materials and methods section. In UM-SCC-3p53mut_n cells, the most significant change in protein expression was noted for p21CIP1/WAF1, which decreased with rising CDDP levels and even more so if MK571 was present. A similar tendency could also be observed for p53. Although not reaching significance, in UM-SCC-3p53mut_n cells, the expression of uncleaved caspase 3 went down in the presence of MK571. This effect was much less pronounced in UT-SCC-26Ap53mut_c cells. In UT-SCC-26Ap53mut_c cells, p21CIP1/WAF1 went up significantly if MK571 was added even in the absence of CDDP but significantly decreased with rising CDDP levels, as did cells that did not receive this inhibitor. Similar to p21CIP1/WAF1, p53 also went up significantly in the same cell line after MK571 addition in the absence of CDDP. Interestingly, in UM-SCC-3p53mut_n cells MK571 treatment induced a marked expression of MAP LC3β, a marker of autophagy in a CDDP dose-dependent fashion. The course of MAP LC3β expression to some level paralleled the reduction of p21CIP1/Waf1 and to a lesser extent p53 levels. A minor induction of MAP LC3β was also noted in UT-SCC-26Ap53mut_c cells in the presence of MK571; however, p21CIP1/Waf1 and p53 levels appeared rather induced after inhibitor treatment. The observed changes in MAP LC3β levels did not reach significance although a clear upregulation was seen in single blots (#3). *p<0.05, **p<0.01, ***p<0.001 (TIFF 4079 kb)

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Tonigold, M., Rossmann, A., Meinold, M. et al. A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53mut exhibits ATP-binding cassette transporter upregulation and high glutathione levels. J Cancer Res Clin Oncol 140, 1689–1704 (2014). https://doi.org/10.1007/s00432-014-1727-y

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