Artikel
In vitro and ex vivo analysis of temozolomide resistance in GBM
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Autoren
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Bodo Haas
- Federal Institute of Drugs and Medical Devices, Bonn, Germany
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Marco Timmer
- Department of General Neurosurgery, University Hospital Cologne, Köln, Germany
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Roland Frötschl
- Federal Institute of Drugs and Medical Devices, Bonn, Germany
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Dirk von Mallek
- Federal Institute of Drugs and Medical Devices, Bonn, Germany
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Gabriele Röhn
- Department of General Neurosurgery, University Hospital Cologne, Köln, Germany
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Niels Eckstein
- Federal Institute of Drugs and Medical Devices, Bonn, Germany; Applied Pharmacy, University of Applied Sciences Kaiserslautern, Campus Pirmasens, Pirmasens, Germany
| Veröffentlicht: | 8. Juni 2016 |
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Gliederung
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Objective: Adjuvant GBM chemotherapy is carried out with the alkylating agent Temozolomide (TMZ). The aggressiveness of GBM is mainly based on resistance to radio- and chemotherapy. In order to understand the underlying molecular mechanisms of resistance we analysed the response of the intrinsic resistant GBM cell line U251 to TMZ. Following in vitro experiments we used tumour samples derived from patients and performed a systematic phospho-proteome profiling approach.
Method: GBM cell line U251 was treated with increasing concentrations of TMZ (1 - 5000 µM) and IC50 was determined in cell viability assays. Cells were subsequently treated with 1000 µM TMZ (IC50) for 72 hours and apoptosis was determined by Annexin V/PI FACS analysis. Proteome profiler arrays™ were performed to detect dysregulated MAP-kinases and apoptotic pathways. Tumor samples of secondary GBM were derived from snap frozen tissue and protein was isolated and subjected to Proteome profiler arrays™.
Results: TMZ induced cell death in U251 cells with an IC50 of 1000 µM. This was paralleled by an increase in apoptosis as measured by Annexin V/PI FACS analysis. Subsequent proteome profiling after TMZ treatment revealed that expression of a wide range of pro-apoptotic proteins (e.g. p53, CD95, Pro-Caspase-3) were strongly induced. These findings were in line with phosphorylation/activation of several kinases (e.g. AKT, GSK-3, JNK) after TMZ treatment, which are involved in cell cycle regulation and drug resistance. To link the in vitro results with the clinical situation we isolated protein from secondary GBM before and after TMZ treatment. (Phospho-) proteome analysis of these samples showed that many (phospho-) proteins were significantly regulated in TMZ-resistant recurrent tumors as compared to untreated samples. In contrast to the findings observed after short term TMZ treatment of U251 cells, after various cycles of TMZ over longer period of time, apoptotic proteins tended to be down-regulated (e.g. pro-caspase-3, RAD17) and kinases such as AKT and CREB were less phosphorylated.
Conclusions: Our investigations revealed that short-term TMZ treatment of U251 cells induced apoptotic cell death only at high concentrations what can be attributed to the intrinsic resistance of U251 cells. Interestingly, after long-term treatment using TMZ in recurrent GBM, apoptosis and cell cycle progression appeared to be reduced resembling the resistant state of the tumor. AKT and CREB appear to play a major role in GBM resistance towards TMZ.
Note: Bodo Haas and Marco Timmer contributed equally.
