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, 3 (10), 1112-23

Disulfiram, a Drug Widely Used to Control Alcoholism, Suppresses the Self-Renewal of Glioblastoma and Over-Rides Resistance to Temozolomide

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Disulfiram, a Drug Widely Used to Control Alcoholism, Suppresses the Self-Renewal of Glioblastoma and Over-Rides Resistance to Temozolomide

Joanna Triscott et al. Oncotarget.

Abstract

Glioblastomas (GBM) are associated with high rates of relapse. These brain tumors are often resistant to chemotherapies like temozolomide (TMZ) and there are very few treatment options available to patients. We recently reported that polo-like kinase-1 (PLK1) is associated with the proliferative subtype of GBM; which has the worst prognosis. In this study, we addressed the potential of repurposing disulfiram (DSF), a drug widely used to control alcoholism for the past six decades. DSF has good safety profiles and penetrates the blood-brain barrier. Here we report that DSF inhibited the growth of TMZ resistant GBM cells, (IC90=100 nM), but did not affect normal human astrocytes. At similar DSF concentrations, self-renewal was blocked by ~100% using neurosphere growth assays. Likewise the drug completely inhibited the self-renewal of the BT74 and GBM4 primary cell lines. Additionally, DSF suppressed growth and self-renewal of primary cells from two GBM tumors.These cells were resistant to TMZ, had unmethylated MGMT, and expressed high levels of PLK1. Consistent with its role in suppressing GBM growth, DSF inhibited the expression of PLK1 in GBM cells. Likewise, PLK1 inhibition with siRNA, or small molecules (BI-2536 or BI-6727) blocked growth of TMZ resistant cells. Our studies suggest that DSF has the potential to be repurposed for treatment of refractory GBM.

Figures

Figure 1
Figure 1. DSF inhibits GBM cell growth and self-renewal
(A-B) SF188 cells were treated with 50, 100 or 500 nM DSF and tumor growth was assessed in monolayer or in serial neurosphere assays. (C-D) Adult GBM BT74 and GBM4 cells were treated with 50-500 nM DSF and self-renewal was assessed in neurosphere assays. Microscopy that demonstrates the effect of DSF treatment on BT74 (E) and GBM4 (F) neurosphere growth. Scale bar = 200 um
Figure 2
Figure 2. Freshly isolated GBM cells are sensitive to DSF yet resistant to TMZ
(A) Primary GBM cells referred to aBT001 and aBT003 were isolated from adult patients with GBM. DNA was isolated from the tumors and subjected to MGMT analysis by PCR. In both cases the MGMT promoter was not methylated indicating that the protein would be expressed (M = methylated, UM = unmethylated). (B-C) The growth of aBT001 was unaffected by TMZ however DSF suppressed their growth by as much as 92% with a single treatment. Cell growth was assessed after 72 hrs. D-E) TMZ was ineffective at suppressing self-renewal when aBT003 cells were exposed to the drug. However DSF suppressed self-renewal by 95-98% based on a single exposure. Scale bar = 200 um.
Figure 3
Figure 3. DSF inhibits the expression of PLK1 in pediatric GBM SF188 cells
(A) SF188 cells were exposed to DSF for 24 hrs using DMSO as a solvent control. Protein and transcript levels of PLK1 were assessed using immunoblotting or qRT-PCR. (B-C) PLK1 inhibition with siRNA inhibits SF188 cell growth and induced apoptosis based on PARP and caspase 3 cleavage. Scramble RNA oligo was transfected as a control. The efficacy of PLK1 inhibition on SF188 growth is exemplified in combination with 10 uM TMZ.
Figure 4
Figure 4. DSF inhibits the expression of PLK1 in adult GBM U251 cells
(A) U251 cells were exposed to DSF for 24 hrs using DMSO as a solvent control. Protein and transcript levels of PLK1 were assessed using immunoblotting or qRT-PCR. (B-C) Inhibiting PLK1 with BI-2536 or siRNA inhibits their growth. The efficacy of PLK1 inhibition on U251 growth is exemplified in combination with 10 uM TMZ. (D) Likewise, DSF inhibits the growth of U251 cells in a dose-dependent manner using DMSO as a solvent control.
Figure 5
Figure 5. Targeting PLK1 inhibits growth of drug resistant cells with upregulated PLK1 protein
(A) U251 cells have lower PLK1 transcript expression than TMZ resistant SF188 cells. U251 cells were treated with 10 uM TMZ every 2 days for a total of 7 days and (B) partial TMZ sensitivity is demonstrated in a growth assay. (C) Immunoblot demonstrating an increase in PLK1 protein levels in TMZ treated U251 cells compared to untreated and DMSO after 24 hours. Actin is used as a loading control protein. (D) The surviving TMZ resistant cells were re-plated and treated with increasing concentrations of BI-2536 for 5 days.
Figure 6
Figure 6. PLK1 inhibitors can be used to over-come TMZ resistance
(A) BT74 cells are resistant to TMZ yet sensitive to PLK1 inhibition with BI-2536. (B) BT241 cells are a second example to which the cells are TMZ resistant yet sensitive to PLK1 inhibition. Both models are maintained as primary isolates and only cultured as neurospheres. The combination of TMZ and BI-2536 did not further improve self-renewal inhibition. Scale bar = 500 um.
Figure 7
Figure 7. aBT001 and aBT003 express high levels of PLK1
(A) PLK1 levels were assessed in aBT001 and aBT003 by immunostaining. Both cases express high levels of PLK1. (B-C) The PLK1 inhibitor BI-2536 suppressed the growth of aBT001 and induced cell death.

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