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Acquired resistance to temozolomide in glioma cell lines: molecular mechanisms and potential translational
Jihong Zhang1, Malcolm F G Stevens, Charles A Laughton
1Centre for Biomolecular Science, School of Pharmacy, University of Nottingham, Nottingham, UK.
Abstract:
Treatment for glioblastoma multiforme includes the alkylating agent temozolomide combined with ionizing radiation. Persistent O6-guanine methylation by temozolomide in O6-methylguanine methyl transferase negative tumors causes cytotoxic lesions recognized by DNA mismatch repair, triggering apoptosis. Resistance (intrinsic or acquired) presents obstacles to successful temozolomide treatment, limiting drug efficacy and life expectancy. Two glioma cell lines, SNB19 and U373, sensitive to temozolomide (GI(50) values 36 and 68 microM, respectively) were exposed to increasing temozolomide concentrations (1-100 microM). Variant cell lines (SNB19VR, U373VR) were generated that displayed acquired temozolomide resistance (GI(50) values 280 and 289 microM, respectively). Cross-resistance to mitozolomide was observed in U373VR cells only. In clonogenic and MTT assays, methylguanine methyltransferase (MGMT) depletion using O6-benzylguanine sensitized U373VR cells to temozolomide, indicating the resistance mechanism involves MGMT re-expression. Indeed, Western blot analyses revealed MGMT protein in cell lysates. In SNB19VR cells, down-regulation of MSH6 message and protein expression may confer temozolomide tolerance. Inhibition of poly(ADP-ribose) polymerase-1 (a key base excision repair (BER) enzyme) partially restored sensitivity, and DNA repair gene arrays demonstrated up-regulation (>5-fold) of BER gene NTL1 in SNB19VR cells. In conclusion, we have developed two glioma cell lines whose distinct mechanisms of acquired resistance to temozolomide, involving expression of MGMT, or inactivation of DNA mismatch repair and recruitment of BER enzymes, are consistent with clinical observations. These cell lines provide valuable models for the development of strategies to combat temozolomide resistance.
Insights
Researchers developed new glioblastoma cell lines to study resistance to temozolomide (TMZ). These models reveal distinct resistance mechanisms involving O6-methylguanine methyltransferase (MGMT) and DNA repair pathways, aiding in developing new treatment strategies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Glioblastoma multiforme (GBM) treatment involves temozolomide (TMZ) and radiation.
- Drug resistance to TMZ limits treatment efficacy and patient survival.
- Understanding resistance mechanisms is crucial for improving GBM therapy.
Purpose of the Study:
- To develop and characterize novel glioma cell lines with acquired resistance to temozolomide.
- To elucidate the distinct molecular mechanisms underlying temozolomide resistance in these cell lines.
- To provide valuable models for investigating strategies to overcome temozolomide resistance.
Main Methods:
- Generation of temozolomide-resistant glioma cell lines (SNB19VR, U373VR) from sensitive parental lines (SNB19, U373).
- Assessment of drug sensitivity using GI(50) values, clonogenic, and MTT assays.
- Analysis of O6-methylguanine methyltransferase (MGMT) expression via Western blot.
- Investigation of DNA repair pathways including mismatch repair (MSH6) and base excision repair (BER) gene expression (NTL1).
- Evaluation of poly(ADP-ribose) polymerase-1 (PARP-1) inhibition effects on drug sensitivity.
Main Results:
- Acquired resistance to temozolomide was successfully established in SNB19VR and U373VR cell lines.
- U373VR cells showed cross-resistance to mitozolomide, linked to MGMT re-expression.
- MGMT depletion sensitized U373VR cells to temozolomide, confirming MGMT's role in resistance.
- SNB19VR cells exhibited MSH6 down-regulation, suggesting DNA mismatch repair involvement.
- Inhibition of PARP-1 partially restored sensitivity in SNB19VR cells, with up-regulation of BER gene NTL1.
Conclusions:
- Two distinct mechanisms of acquired temozolomide resistance in glioma cells were identified: MGMT re-expression and alterations in DNA repair pathways (mismatch repair and base excision repair).
- These developed cell lines serve as robust models for studying temozolomide resistance in glioblastoma.
- The findings support the development of targeted strategies to overcome drug resistance and improve glioblastoma treatment outcomes.
