Revealing the epigenetic effect of temozolomide on glioblastoma cell lines in therapeutic conditions

Abstract

Temozolomide (TMZ) is a drug of choice in glioblastoma treatment. Its therapeutic applications expand also beyond high grade gliomas. However, a significant number of recurrences and resistance to the drug is observed. The key factor in each chemotherapy is to achieve the therapeutic doses of a drug at the pathologic site. Nonetheless, the rate of temozolomide penetration from blood to cerebrospinal fluid is only 20-30%, and even smaller into brain intestinum. That makes a challenge for the therapeutic regimens to obtain effective drug concentrations with minimal toxicity and minor side effects. The aim of our research was to explore a novel epigenetic mechanism of temozolomide action in therapeutic conditions. We analyzed the epigenetic effects of TMZ influence on different glioblastoma cell lines in therapeutically achieved TMZ concentrations through total changes of the level of 5-methylcytosine in DNA, the main epigenetic marker. That was done with classical approach of radioactive nucleotide post-labelling and separation on thin-layer chromatography. In the range of therapeutically achieved temozolomide concentrations we observed total DNA hypomethylation. The significant hypermethylating effect was visible after reaching TMZ concentrations of 10-50 μM (depending on the cell line). Longer exposure time promoted DNA hypomethylation. The demethylated state of the glioblastoma cell lines was overcome by repeated TMZ applications, where dose-dependent increase in DNA 5-methylcytosine contents was observed. Those effects were not seen in non-cancerous cell line. The increase of DNA methylation resulting in global gene silencing and consecutive down regulation of gene expression after TMZ treatment may explain better glioblastoma patients' survival.

Fig 1. The main reaction products of the TMZ reaction with DNA.

7-methylguanosine (m⁷G) is the most abundant product (60–80%) of TMZ reaction with DNA, followed by 3-methyladenosine (m³A, 10–20%) and O6-methylguanosine (O⁶mG, 5%). Other products constitute ca. 15% [17].

Fig 2. A flow chart of the total DNA methylation (m⁵C) analysis.

Isolated DNA is hydrolyzed to 3’-mononucleotides (Np: A/adenosine, G/guanosine, C/cytidine, T/thymidine). The hydrolysate is labelled with [γ-³²P] ATP, dephosphorylated (detachment of 3’ phosphate) and separated with two-dimensional TLC. The chromatogram is then evaluated with phosphoimager and the spots’ intensities are measured. Those values are used for the calculation of the R coefficient according to the given equation [27].

Fig 3. The effect of TMZ on total DNA methylation of T98G cell line.

The analysis was performed after 3 (white bars), 12 (light grey bars), 24 (dark grey bars) and 48 (black bars) hrs of incubation in a given TMZ concentration (0.5–100 μM). Control cells (C) were treated with DMSO only. The R values are means from three experiments ±SD. Asterisks indicate a significant difference (*p < 0,05, **p < 0,01, ***p < 0,001) from the control (DMSO) value. Striking DNA hypomethylation happens in therapeutically achieved concentrations (less than 5 μM). Significant DNA hypermethylation is achieved at minimal TMZ concentrations of 20–50 μM in 3–24 hrs incubation time. Clear demethylating tendency is seen with 48 hrs incubation time in all TMZ concentrations.

Fig 4. The effect of TMZ on total DNA methylation of U138 cell line.

The analysis was performed after 3 (white bars), 12 (light grey bars), 24 (dark grey bars) and 48 (black bars) hrs of incubation in a given TMZ concentration (0.5–100 μM). Control cells (C) were treated with DMSO only. The R values are means from three experiments ±SD. Asterisks indicate a significant difference (*p < 0,05, **p < 0,01, ***p < 0,001) from the control (DMSO) value. A very little DNA hypermethylating effect is observed (for incubation times 3 and 12 hrs, over concentrations 20 and 30 μM respectively). Very clear initial hypomethylation is seen in TMZ concentrations up to 100 μM for incubation times of 24 and 48 hrs.

Fig 5. The effect of TMZ on total DNA methylation of U118 cell line.

The analysis was performed after 3 (white bars), 12 (light grey bars), 24 (dark grey bars) and 48 (black bars) hrs of incubation in a given TMZ concentration (0.5–100 μM). Control cells (C) were treated with DMSO only. The R values are means from three experiments ±SD. Asterisks indicate a significant difference (*p < 0,05, **p < 0,01, ***p < 0,001) from the control (DMSO) value. Significant demethylating tendency is seen with 48 hrs incubation time in all TMZ concentrations. The TMZ amount needed to reach hypermethylating effect depends on incubation time and is within the range 1–10 μM.

Fig 6. The effect of TMZ on total DNA methylation of HaCaT cell line.

The analysis was performed after 3 (white bars), 12 (light grey bars), 24 (dark grey bars) and 48 (black bars) hrs of incubation in a given TMZ concentration (0.5–100 μM). Control cells (C) were treated with DMSO only. The R values are means from three experiments ±SD. Asterisks indicate a significant difference (*p < 0,05, **p < 0,01, ***p < 0,001) from the control (DMSO) value. In all incubation times the hypermethylation effect is seen over TMZ concentration of 5 μM. No hypomethylation in lower concentrations was detected.

Fig 7. The effect of repetitive doses of TMZ on total DNA methylation.

Total amount of m⁵C in DNA (R) changes during Stupp-mimicking TMZ-therapy. TMZ was administered to glioblastoma (T98G –white bars, U138 –light grey bars, U118 –dark grey bars) and keratinocyte (HaCaT–diagonally striped bars) cell lines at 0.5, 1, 3, 5, 10 μM concentrations for 5 days. Each day cells were washed with PBS and placed in fresh medium with indicated TMZ concentrations. Control (C) cells were treated with DMSO only. The total DNA methylation was estimated after 5^th day of treatment. The R values are means from three experiments ±SD. Asterisks indicate a significant difference (*p < 0,05, **p < 0,01, ***p < 0,001) from the control (DMSO) value.

Fig 8. The impact of TMZ exposure period on DNA methylation in T98G cell line.

T98G cell culture was treated with the given single dose of TMZ (0.5, 1, 3, 5, 10 μM) and left for 1–7 days. The global DNA methylation was estimated after 1^st, 2^nd, 3^rd, 4^th, 5^th, and 7^th day of incubation. The highest methylation (R) is seen on day 1 in all concentrations. In concentrations over 3 μM we observe increasing DNA methylation. On day 7 a hypomethylating trend is observed potentiated by increasing concentration.

Fig 9. The impact of TMZ exposure period on DNA methylation in U138 cell line.

U138 cell culture was treated with the given single dose of TMZ (0.5, 1, 3, 5, 10 μM) and left for 1–7 days. The global DNA methylation was estimated after 1^st, 2^nd, 3^rd, 4^th, 5^th, and 7^th day of incubation. The hypomethylation at prolonged time (7 days) is clearly seen. The hypermethylation is achieved in shorter incubation times in concentrations over 5 μM.

Fig 10. The impact of TMZ exposure period on DNA methylation in U118 cell line.

U118 cell culture was treated with the given single dose of TMZ (0.5, 1, 3, 5, 10 μM) and left for 1–7 days. The global DNA methylation was estimated after 1^st, 2^nd, 3^rd, 4^th, 5^th, and 7^th day of incubation. A general hypermethylation is observed, with no initial hypomethylating effect in lower concentration range. Hypomethylation after 7 days starts with concentrations over 1 μM.

Fig 11. The impact of TMZ exposure period on DNA methylation in HaCaT cell line.

HaCaT cell culture was treated with the given single dose of TMZ (0.5, 1, 3, 5, 10 μM) and left for 1–7 days. The global DNA methylation was estimated after 1^st, 2^nd, 3^rd, 4^th, 5^th, and 7^th day of incubation. No significant change in DNA methylation is observed during time.