Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells

Abstract

Background: Glioblastoma multiforme (GBM) cells are resistant to anticancer drugs. Cancer stem cells (CSCs) are a key mediator of chemoresistance. We have reported that disulfiram (DS), an aldehyde dehydrogenase (ALDH) inhibitor, targets breast CSC-like cells. In this study, the effect of DS and combination of DS and gemcitabine (dFdC) on GBM cells and GBM stem-like cells was investigated.

Methods: 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)-isobologram, western blot, luciferase reporter gene assay, electrophoretic mobility-shift assay and ALDH analysis were used in this study.

Results: Disulfiram is cytotoxic in GBM cell lines in a copper (Cu)-dependent manner. Disulfiram/copper enhances the cytotoxicity of dFdC. Combination index-isobologram analysis indicates a synergistic effect between DS/Cu and dFdC. Disulfiram/copper induces reactive oxygen species (ROS), activates JNK and p38 pathways and inhibits nuclear factor-kappa B activity in GBM cell lines. Disulfiram/copper may trigger intrinsic apoptotic pathway via modulation of the Bcl2 family. Disulfiram/copper abolishes stem-like cell population in GBM cell lines.

Conclusion: Our findings indicate that the cytotoxicity of DS/Cu and the enhancing effect of DS/Cu on the cytotoxicity of dFdC in GBM stem-like cells may be caused by induction of ROS and inhibition of both ALDH and the NFkB pathway. Both DS and dFdC can traverse the blood-brain barrier. Further study may lead them into GBM chemotherapy.

Figure 1

Disulfiram/copper-induced apoptosis in GBM cell lines. (A) Annexin V assay. The GBM cells were exposed to 0.5 μℳ of disulfiram (DS), 1 μℳ of CuCl₂ (Cu), 1 μℳ of gemcitabine (dFdC) or disulfiram plus CuCl2 (DS/Cu) and disulfiram plus CuCl2 plus gemcitabine (DS/Cu/dFdC) in combination for 48 h. The drug-treated and control cells (2 × 10⁵) were stained with PI and Annexin V and analysed by flow cytometry. (B) The percentage of different cell populations identified by PI/Annexin V assay. **P<0.01 (Mean+s.d.; n=3); (C) The expression levels of cleaved PARP, Bax and Bcl2 protein. The expression of cleaved PARP, Bax and Bcl2 proteins were analysed by western blot in GBM cell lines exposed to 0.5 μℳ of DS, 1 μℳ of Cu or 0.5 μℳ of DS plus 1 μℳ of Cu for 48 h. Tub: α-tubulin used as a loading control. (D) The ratio of Bax/Bcl2. The band density in C was analysed by ImageJ programme. Abbreviations: Nec=necrosis; Apo=apoptosis.

Figure 2

Reactive oxygen species (ROS) activity is related to DS/Cu-induced cytotoxicity. (A, B) Disulfiram/copper induces ROS generation. The cancer cells (2 × 10⁵ cells per well) were exposed to DS (250 nℳ), Cu (1 μℳ), DS (250 nℳ) plus Cu (0.5–1 μℳ) for 5–30 min. Hydrogen peroxide (100 μℳ) and pyocyanin (200 μℳ) were used as positive control. (A) The dot lines represent the untreated cells and the solid lines represent DS-, Cu-, DS/Cu- and pyocyanin-treated cells, respectively. In NAC-treated group, the dot and solid lines represent DS/Cu-treated cells with or without NAC reversion, respectively. (B) The ROS activity in DS/Cu and H₂O₂-treated cells was reversed by co-exposure to NAC (2 mℳ). Non-treated cells are denoted by –VE. (C) The effect of ROS and MAPK pathway inhibitors on DS/Cu-induced cytotoxicity. The cancer cells were exposed to DS (500 nℳ)/Cu (500 nℳ), DS/Cu plus NAC (2 mℳ), SP600125 (10 μℳ) or SB203580 (10 μℳ) for 72 h. The viability of drug-treated cells was determined by MTT assay. Hydrogen peroxide (100 μℳ)-treated group was used as a control.

Figure 3

Disulfiram/copper activated MAPK pathway and inhibited NFκB activity. (A–C) The GBM cells were exposed to dFdC_1 μℳ, DS_1 μℳ/Cu_1 μℳ or dFdC_1 μℳ/DS_1 μℳ/Cu_1 μℳ for 3, 6 and 24 h. The expression levels and phosphorylation status of proteins in JNK pathway were examined by western blot. (D) Disulfiram/copper activates p38 pathway. The GBM cell lines were exposed to different treatments for 24 h. The phosphorylation status of p38 was examined by western blot. (E) The effect of DS, Cu alone or DS and Cu in combination (DS/Cu) on phosphorylation status of c-Jun and ERK. To rule out the influence of trace amount of copper contained in the serum, the GBM cell lines were cultured in serum-free medium containing DS_1 μℳ or Cu_1 μℳ alone or DS_1 μℳ/Cu_1 μℳ for 24 h. The phosphorylation of c-Jun and ERK was detected by western blot. Alpha-tubulin (TUB) was used as a loading control. (F) N-acetyl-cysteine inhibited DS/Cu-induced c-Jun and p38 phosphorylation. The GBM cell lines were exposed to DS_1 μℳ/Cu_1 μℳ with or without NAC (2 mℳ) for 24 h. The phosphorylation status of c-Jun and p38 was examined by western blot. (G) Electrophoretic mobility-shift assay analysis of NFκB DNA binding activity. Nu: western blot of nucleolin was used as a protein loading control. (H) Examination of NFκB transcriptional activity by luciferase reporter gene assay. The GBM cell lines were exposed to dFdC_1 μℳ, DS_1 μℳ/Cu_1 μℳ or dFdC_1 μℳ/DS_1 μℳ/Cu_1 μℳ for 24 h before subjected to EMSA and luciferase reporter gene assay.

Figure 4

The effect of DS/Cu on stem-like status in GBM cell lines. (A) Disulfiram/copper and DS/Cu/dFdC blocked neurosphere-forming ability in GBM cell lines. The GBM cells formed neurospheres after 7 days culture in SCM. The neurosphere cells were separated by tripsinization and exposed to DS_0.5 μℳ, Cu_1 μℳ, dFdC_1 μℳ alone or DS_0.5 μℳ/Cu_1 μℳ and DS_0.5 μℳ/Cu_1 μℳ/dFdC_1 μℳ for 3 h. The drug-treated cells were cultured at a cell density of 20 cells per well in 30 wells of ultra-low attachment 96-well plates containing drug-free SCM for 14 days. The neurospheres were photographed at × 40 magnificaiton. (B) Disulfiram/copper and DS/Cu/dFdC abolished ALDH-positive population in neurospheres. The neurosphere cells were treated as those in (A). The ALDH-positive cells were determined by ALDEFLUOR staining and flow cytometer analysis. (C) Aldehyde dehydrogenase activity in neurosphere cells were not directly inhibited by DS/Cu. Diethylaminobenzaldehyde (DEAB, 30 μℳ), DS (0.5 μℳ) and DS (0.5 μℳ) plus Cu (1 μℳ) were added into the ALDH staining tubes and incubated at 37 °C for 30 min. The ALDH activity in the cells was detected by flow cytometer. The inserted numbers represent percentage of the positive cells (mean (s.d.), n=3). **P<0.01 (in comparison with NS_−VE), ^¶P<0.01 (in comparison with other groups). Abbreviation: NS=neurosphere.