Clinical and Translational Oncology

, Volume 20, Issue 4, pp 508–516 | Cite as

Acquired temozolomide resistance in human glioblastoma cell line U251 is caused by mismatch repair deficiency and can be overcome by lomustine

  • J. Stritzelberger
  • L. Distel
  • R. Buslei
  • R. Fietkau
  • F. Putz
Research Article

Abstract

Purpose

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. While the alkylating agent temozolomide (TMZ) has prolonged overall survival, resistance evolution represents an important clinical problem. Therefore, we studied the effectiveness of radiotherapy and CCNU in an in vitro model of acquired TMZ resistance.

Methods

We studied the MGMT-methylated GBM cell line U251 and its in vitro derived TMZ-resistant subline, U251/TMZ-R. Cytotoxicity of TMZ, CCNU, and radiation was tested. Both cell lines were analyzed for MGMT promotor status and expression of mismatch repair genes (MMR). The influence of MMR inhibition by cadmium chloride (CdCl2) on the effects of both drugs was evaluated.

Results

During the resistance evolution process in vitro, U251/TMZ-R developed MMR deficiency, but MGMT status did not change. U251/TMZ-R cells were more resistant to TMZ than parental U251 cells (cell viability: 92.0% in U251/TMZ-R/69.2% in U251; p = 0.032) yet more sensitive to CCNU (56.4%/80.8%; p = 0.023). The effectiveness of radiotherapy was not reduced in the TMZ-resistant cell line. Combination of CCNU and TMZ showed promising results for both cell lines and overcame resistance. CdCl2-induced MMR deficiency increased cytotoxicity of CCNU.

Conclusion

Our results confirm MMR deficiency as a crucial process for resistance evolution to TMZ. MMR-deficient TMZ-resistant GBM cells were particularly sensitive to CCNU and to combined CCNU/TMZ. Effectiveness of radiotherapy was preserved in TMZ-resistant cells. Consequently, CCNU might be preferentially considered as a treatment option for recurrent MGMT-methylated GBM and may even be suitable for prevention of resistance evolution in primary treatment.

Keywords

Glioblastoma Antineoplastic drug resistance Temozolomide Lomustine Mismatch repair 

Abbreviations

7AAD

7-Amino-actinomycin D

AxV

Annexin V

CCNU

Lomustine

CdCl2

Cadmiumchloride

DMEM

Dulbecco’s Modified Eagle Medium (Medium für Zellkultur)

GBM

Glioblastoma multiforme

Gy

Gray

MD

Mean difference

MGMT

O6-Methylguanin-DNA-methyltransferase

MMR

Mismatch-repair

SF

Survival fraction

TMZ

Temozolomide

Notes

Acknowledgements

We would like to thank Mrs. Elisabeth Müller, Mrs. Doris Mehler, and Mrs. Renate Siebert for their technical expertise and help in conducting experiments. The present work was performed in fulfillment of the requirements for obtaining the degree “Dr. Med.”

Author contributions

JS and FP performed the experiments mentioned in the “Materials and methods”, except for the pyrosequencing. RB performed pyrosequencing. JS, FP, LD were major contributors in writing the manuscript. RF provided materials and working space for the experiments we performed and revised the manuscript critically. All authors read and approved the final manuscript.

Compliance with ethical standards

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest

The authors declare that they have no competing interests.

Funding

No funding was received.

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Copyright information

© Federación de Sociedades Españolas de Oncología (FESEO) 2017

Authors and Affiliations

  1. 1.Department of Radiation OncologyFriedrich-Alexander-University Erlangen-Nürnberg (FAU)ErlangenGermany
  2. 2.Department of NeuropathologyFriedrich-Alexander-University Erlangen-Nürnberg (FAU)ErlangenGermany

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