INTRODUCTION: Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a very poor prognosis. The standard treatment for GBM is surgical resection followed by chemoradiotherapy with temozolomide (TMZ), an alkylating agent which methylates the DNA at various sites. Methylation of the guanin at the O6 position leads to a falsely inserted base nucleotide during transcription, which subsequently activates miss match repair (MMR), resulting in numerous single strand breaks and finally in cell death. Although TMZ extends the survival rate, tumors rapidly develop resistance, which is mainly based on the O6-methylguanine-DNA-methyltransferase (MGMT) expression as well as base excision repair (BER) and MMR. Clinical studies show that low expression of MGMT in glioma patients resulted in a better therapeutical outcome. To increase TMZ efficiency in GBM, a combined treatment with highly specific MGMT inhibitors like Lomeguatrib (LG) is used.

MATERIALS AND METHODS: We studied both a commercially available TMZ resistant (T98G) and non-resistant (U373) glioblastoma cell line as well as the resistant cell line U373R which was generated by us via long term TMZ treatment of U373. Normal human dermal fibroblasts (NHDF) were used as control for normal (healthy) cells. All cells were treated with increasing concentrations of TMZ from 10 µM to 1000 µM for 48 to 96h and/or 50 µM LG, respectively. Cell viability was determined by MTT and SRB Assay. MGMT occurrence was examined via western blot and qPCR. Incubation with 10 µM 5-Aza-2´-desoxycytidine (Decitabin) for 5 days was used to determine the effect of the that methylation inhibitor on MGMT expression.

RESULTS AND DISCUSSION: U373 cells show a significant decrease in cell viability after TMZ treatment compared to U373-R and T98G. For T98G, this result can be explained by a generally high expression of MGMT. Surprisingly, the U373R cells showed resistance from TMZ treatment without showing any MGMT expression. The use of the DNA-hypomethylating agent Decitabin indicates an involvement of MGMT promotor hypermethylation in the observed downregulation of MGMT in U373R. To find out whether the MMR is also involved in the observed TMZ resistance, qPCR was performed to check for basal expression of MMR subunits, and indeed, a lowered expression of the 2 subunits MSH2 and MSH6, being major players in MMR, was observed in U373R compared to U373 and T98G.

CONCLUSION: The observed data verify that there are different resistance mechanisms in GBM cells to overcome TMZ toxicity. Although it is known that other resistance mechanisms apart from MGMT overexpression exist in GBM, it was shown for the first time that long term treatment with TMZ results in a loss of MGMT instead of an increase in expression. Those mechanisms must be evaluated to get a better understanding of the origin of the resistance, and these data may support the development of new treatment strategies.