Cancer Res Treat.  2019 Apr;51(2):696-705. 10.4143/crt.2018.249.

Disulfiram, a Re-positioned Aldehyde Dehydrogenase Inhibitor, Enhances Radiosensitivity of Human Glioblastoma Cells In Vitro

• Koh HK
• Seo SY
• Kim JH
• Kim HJ
• Chie EK
• Kim SK
• Kim IH

• KMID: 2464415
• DOI: http://doi.org/10.4143/crt.2018.249

Abstract

PURPOSE
Glioblastoma, the most common brain tumor in adults, has poor prognosis. The purpose of this study was to determine the effect of disulfiram (DSF), an aldehyde dehydrogenase inhibitor, on in vitro radiosensitivity of glioblastoma cells with different methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT) promoter and the underlying mechanism of such effect.
MATERIALS AND METHODS
Five human glioblastoma cells (U138MG, T98G, U251MG, U87MG, and U373MG) and one normal human astrocyte (NHA) cell were cultured and treated with DSF or 6MV X-rays (0, 2, 4, 6, and 8 Gy). For combined treatment, cells were treated with DSF before irradiation. Surviving fractions fit from cell survival based on colony forming ability. Apoptosis, DNA damage repair, and cell cycle distributionwere assayed bywestern blot for cleaved caspase-3, γH2AX staining, and flow cytometry, respectively.
RESULTS
DSF induced radiosensitization in most of the glioblastoma cells, especially, in the cells with radioresistance as wildtype unmethylated promoter (MGMT-wt), but did not in normal NHA cell. DSF augmented or induced cleavage of caspase-3 in all cells after irradiation. DSF inhibited repair of radiation-induced DNA damage in MGMT-wt cells, but not in cells with methylated MGMT promoter. DSF abrogated radiation-induced G2/M arrest in T98G and U251MG cells.
CONCLUSION
Radiosensitivity of glioblastoma cells were preferentially enhanced by pre-irradiation DSF treatment compared to normal cell, especially radioresistant cells such as MGMT-wt cells. Induction of apoptosis or inhibition of DNA damage repair may underlie DSF-induced radiosensitization. Clinical benefit of combining DSF with radiotherapy should be investigated in the future.

Keyword

Disulfiram; Glioblastoma; MGMT; Radiosensitivity; DNA damage repair; Apoptosis

MeSH Terms

Adult
Aldehyde Dehydrogenase*
Apoptosis
Astrocytes
Brain Neoplasms
Caspase 3
Cell Cycle
Cell Survival
Disulfiram*
DNA Damage
Flow Cytometry
Glioblastoma*
Humans*
In Vitro Techniques*
Methylation
Prognosis
Radiation Tolerance*
Radiotherapy
Aldehyde Dehydrogenase
Caspase 3
Disulfiram

Figure

• 

  Fig. 1. Expression of O6-methylguanine-DNA methyltransferase (MGMT) protein in glioblastoma cells. U138-MG and T98G cells expressed MGMT whereas U251MG, U87MG, or U373MG cells did not express MGMT based on western blots. After disulfiram (DSF) treatment for 24 hours, U138MG and T98G cells maintained expression of MGMT.

• 

  Fig. 2. (A-F) Cell survival curves fitting to a linear-quadratic model after irradiation or pre-irradiation disulfiram (DSF) treatment. Radiosensitivity of glioblastoma cells was enhanced by pre-irradiation DSF treatment except for U373MG (E) or normal human astrocyte (NHA) (F). Enhancement was especially pronounced in U138MG (A) and T98G (B) cells with wildtype unmethylated O6 -methylguanine-DNA methyltransferase promoter. Each experiment was repeated at least three times with similar results. IR, irradiation.

• 

  Fig. 3. Sensitizer enhancement ratio at 0.5 (SER0.5). (A) Mean SER0.5 value±standard deviation (SD) in all cells. Wildtype unmethylated O6-methylguanine-DNA methyltransferase (MGMT) promoter (MGMT-wt) cells and methylated MGMT promoter (MGMT-meth) cells are indicated. (B) The relationship between survival fraction at 2 Gy (SF2) and SER0.5 was illustrated in log scale. Error bars indicated SD. There was an inverse relationship between radiosensitivity and the degree of disulfiram-induced enhancement of radiosensitivity in glioblastoma cells.

• 

  Fig. 4. Western blot analysis of cleaved caspase-3 after 0, 2, 6, and 24 hours of irradiation (IR). Pre-irradiation disulfiram (DSF) either induced expression of cleaved caspase-3 in U138MG, U87MG, and U373MG cells or augmented it in T98G and U251MG cells.

• 

  Fig. 5. γH2AX assay. Anti-γH2AX antibody was applied and then fluorescein isothiocyanate–labeled secondary antibody was added (arrowheads). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, arrows). (A, B) γH2AX foci after irradiation (IR) were more prevalent in pre-irradiation disulfiram (DSF) treatment group than those in IR (6 Gy) alone group until 24 hours in U138MG and T98G cells with wildtype unmethylated O6-methylguanine-DNA methyltransferase promoter (×1,000). Scale bars=10 μm. (C) The decrease in proportion of γH2AX positive cells by the time after IR was significantly blocked by pre-irradiation DSF in U138MG and T98G cells, but was not in U251MG, U87MG, or U373MG cells. *p < 0.05.

• 

  Fig. 6. Proportion of cells at G2/M phase and sub-G1 fraction by flowcytometry. (A) Disulfiram (DSF) abrogated irradiation (IR)-induced G2/M arrest in T98G and U251MG cells. (B) DSF failed to affect IR-induced change in sub-G1 fraction. Con, control.

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