Effects of Long-Term Temozolomide Treatment on Glioblastoma and Astrocytoma WHO Grade 4 Stem-like Cells

Abstract

Glioblastoma leads to a fatal course within two years in more than two thirds of patients. An essential cornerstone of therapy is chemotherapy with temozolomide (TMZ). The effect of TMZ is counteracted by the cellular repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT). The MGMT promoter methylation, the main regulator of MGMT expression, can change from primary tumor to recurrence, and TMZ may play a significant role in this process. To identify the potential mechanisms involved, three primary stem-like cell lines (one astrocytoma with the mutation of the isocitrate dehydrogenase (IDH), CNS WHO grade 4 (HGA)), and two glioblastoma (IDH-wildtype, CNS WHO grade 4) were treated with TMZ. The MGMT promoter methylation, migration, proliferation, and TMZ-response of the tumor cells were examined at different time points. The strong effects of TMZ treatment on the MGMT methylated cells were observed. Furthermore, TMZ led to a loss of the MGMT promoter hypermethylation and induced migratory rather than proliferative behavior. Cells with the unmethylated MGMT promoter showed more aggressive behavior after treatment, while HGA cells reacted heterogenously. Our study provides further evidence to consider the potential adverse effects of TMZ chemotherapy and a rationale for investigating potential relationships between TMZ treatment and change in the MGMT promoter methylation during relapse.

Keywords: IDH; MGMT; astrocytoma; cancer stem cells; glioblastoma; temozolomide; therapy.

Figure 1

Glioblastoma and HGA stem-like glioma-initiating cells stained positive for SOX-2 and Nestin. Exemplary immunofluorescence staining of SOX-2 (red), Nestin (green), and DAPI (blue) in pre-treatment MGMT+ (a), post-5-days/-23 days DMSO treatment HGA (b), and monolayer cell cultures and 6 weeks of TMZ treatment MGMT+ neurosphere culture (c). The scale bars represent 100 µm each. Abbreviations: HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; DAPI, 4′,6-Diamidin-2-phenylindol; MGMT+, glioblastoma cell line with >50% O⁶-methylguanine-DNA-methyltransferase promoter methylation; and DMSO, dimethyl sulfoxide.

Figure 2

The increased proliferation and migration of TMZ-treated glioblastoma and HGA stem-like glioma-initiating cells was dependent on their MGMT promoter methylation status. An xCELLigence device was used to determine the proliferation and migration rate of MGMT- (a,b), HGA (c,d) and MGMT+ cell lines. The latter represent pooled samples as their long-term treatment had to be discontinued (e,f). After normalizing the values to their respective DMSO controls, the cells’ proliferation (g) and migration rate (h) after the full 8 weeks of therapy and recovery was compared. Abbreviations: HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; MGMT, O⁶-methylguanine-DNA-methyltransferase; MGMT-, the glioblastoma cell line without MGMT promoter methylation; MGMT+, the glioblastoma cell line with high MGMT promoter methylation; TMZ, the sample treated with temozolomide; DMSO, the sample treated with dimethyl sulfoxide; 5 d/23 d, the sample of the 5-day-treatment/23-day-recovery-treatment group; and 6 w, the sample of the 6-week-treatment group.

Figure 3

The MGMT promoter methylation changes of the glioblastoma and the HGA stem-like glioma-initiating cells. The MGMT promoter methylation of the MGMT- (a,b) and HGA (c,d) cell lines was determined 4 and 8 weeks after 5 d/23 d and 6 w treatment scheme initiation, respectively. As MGMT+ cells treated with TMZ had to be pooled; their results are not shown in this bar-graph. Abbreviations: MGMT, O⁶-methylguanine-DNA-methyltransferase; MGMT-, the glioblastoma cell line without the MGMT promoter methylation; HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; TMZ, the sample treated with temozolomide; DMSO, the sample treated with dimethyl sulfoxide; 5 d/23 d, the sample of the 5-day-treatment/23-day-recovery-treatment group; and 6 w, the sample of the 6-week-treatment group.

Figure 4

A comparison of the sensitivity of glioblastoma and HGA stem-like glioma initiating cells to renewed short-term TMZ treatment. The cells’ response to renewed TMZ exposure was tested after the total 8-week-long treatment schemes utilizing an MTT-assay by re-exposing the pretreated cells to TMZ concentrations of 100 µM (a) and 1000 µM (b) for 48 h. The values were normalized to the results of their respective DMSO treatment group. Circles represent outliers. In addition, the metabolic activity of MGMT- (c,d), HGA (e,f), and MGMT+ (g,h) cell lines was determined in comparison to their respective DMSO controls (of the MTT assay). All values were normalized to their positive and negative controls of the MTT-assay. Abbreviations: HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; TMZ, the temozolomide treated sample; DMSO, the sample treated with dimethyl sulfoxide; MTT, 5-(3-methyltriazen-1-yl)-imidazole-4-carbox-amide; MGMT+, glioblastoma cell line with >50% O⁶-methylguanine-DNA-methyltransferase promoter methylation; MGMT-, glioblastoma cell line without O⁶-methylguanine-DNA-methyltransferase promoter methylation; 5 d/23 d, sample of the 5-day-treatment/23-day-recovery-treatment group; 6 w, the sample of the 6-week-treatment group.

Figure 5

The scheme of TMZ and MGMT effects. In non-cancerous, as well as in glioma cells, TMZ introduces alkylating changes at the DNA, such as O⁶meG methylation. In the case of an unmethylated MGMT promoter, the MGMT enzyme is expressed and transfers the methyl-group to itself, repairing the DNA (left). Cells with the methylated MGMT promoter do not or only marginally express MGMT. In this case, the DNA damage persists and may trigger apoptosis or cytotoxicity (right). The schematic diagram was partially created with biorender.com (a). A summary of the experimental results. ↑ = upregulation, ↓ = downregulation (b). Abbreviations: HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; MGMT, O⁶-methylguanine-DNA-methyltransferase; MGMT+, the glioblastoma cell line with >50% MGMT promoter methylation; MGMT-, the glioblastoma cell line without MGMT promoter methylation; O⁶meG, O⁶-methylated Guanine; TMZ, temozolomide; and w, weeks.

Figure 6

The scheme of the experimental setup to determine the behavioral and molecular changes in the glioblastoma and HGA stem-like glioma-initiating cells with different MGMT promoter methylation status. Three glioblastoma stem-like glioma-initiating cell lines (MGMT+, MGMT-, and HGA) were treated with TMZ or DMSO as a control for 8 weeks in two different schemes reflecting the standard of care. Cells were either treated with 2 cycles of DMSO (a) or TMZ (b) given for 5 days followed by 23 days of recovery (5 d/23 d). A second set of cells was treated continuously with DMSO (c) or TMZ (d) for 6 weeks followed by a 2-week recovery (6 w). Due to the strong effects of TMZ, the treatment of the MGMT+ cells had to be discontinued after 39 days (5 d/23 d scheme, (e)) and 32 days (6 w scheme, (f)), respectively. The surviving cells of the experimental repetitions were pooled to secure their survival (e,f). The treatment of the DMSO groups was stopped accordingly; however, individual samples were kept (a,g). The cells’ changes in proliferation, migration, therapeutic response, and molecular characteristics (MGMT promoter methylation status) caused by the TMZ chemotherapy were determined in follow-up experiments. Abbreviations: 5 d/23 d, 5 days/23 days treatment scheme; 6 w, 6 weeks treatment scheme; HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; MGMT+, glioblastoma cell line with >50% O⁶-methylguanine-DNA-methyltransferase promoter methylation; MGMT-, glioblastoma cell line without O⁶-methylguanine-DNA-methyltransferase promoter methylation; TMZ, temozolomide; DMSO, dimethyl sulfoxide; and gDNA, genomic desoxyribonucleic acid.

Figure 7

The exemplary xCELLigence curves to determine the proliferation and migration. The cells’ proliferation was measured by determining the mean doubling time after the adhesion period and while the cell index displayed the characteristic constant linear slope (a). Similarly, the cells’ migration rate was determined in CIM-plates by measuring the cell index’s slope in a timeframe after the adhesion period had taken place and before cells reached a linear constant slope resembling proliferation (b).