Copper Gluconate/Disulfiram

Cost-effective management of Urinary Bladder Cancer (UBC) is an unmet need

Our study aims to demonstrate the efficacy of a drug repurposing strategy by using disulfiram (DSF) and copper gluconate (Cu) as an add-on treatment combination to traditional GC-based chemother-apy against N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced UBC mice (C57J) model.

Male C57BL/6J mice were given 0.05% BBN in drinking water ad libitum, and tumour for-mation was verified by histological and physical evaluation. Animals were subsequently divided into eight groups and received treatment with different drug combinations. Control animals received only ve-hicle (DMSO). At the end of the treatment schedule, the bladder tumour was excised and further used to check the expression (mRNA and protein) of ALDH1 isoenzymes using qRT-PCR, western blot, and IHC methods. Autophagy induction was assessed by quantifying the expression of LC3B and SQSTM1/p62 proteins through IHC. Biochemical analysis of superoxide dismutase (SOD), reduced glutathione (GSH), and lipid peroxidation levels in the freshly isolated tumours was performed to check the alterations in the antioxidant system caused by combination treatment.

We observed significant induction of an invasive form of bladder cancer in the mice after nine-teen weeks of BBN exposure. The animals began exhibiting early indications of inflammatory alterations as early as the sixth week following BBN treatment. Furthermore, the wet bladder weight and overall tu-mour burden were significantly decreased (p< 0.0001) by DSF-Cu co-treatment in addition to the GC-based chemotherapy. Real-time PCR analysis revealed that treatment with disulfiram and copper glu-conate significantly decreased (p<0.0001) the mRNA expression of ALDH1 isoenzymes. Comparing the triple drug combination group (GC+DSF-Cu) to the untreated mice, a significant rise in LC3B puncta (p<0.0001) and a decrease in P62/SQSTM1 (p=0.0002) were noted, indicating the induction of autophagy flux in the add-on group. When GC+DSF-Cu treated mice were compared to the untreated tumour group, a substantial decrease in ALDH1/2 protein expression was observed (p= 0.0029 in IHC and p<0.0001 in western blot). Lipid peroxidation was significantly higher (p<0.0001) in the triple drug combination group than in untreated mice. There was a simultaneous decrease in reduced glutathione (GSH) and en-zyme superoxide dismutase (SOD) levels (p<0.0001), which strongly suggests the generation of reactive oxygen species and induction of ferroptotic cell death in the add-on therapy group. Additionally, in both IHC and western blot assays, ALDH1A3 expression was found to be significantly increased (p=0.0033, <0.0001 respectively) in GC+DSF-Cu treated mice relative to the untreated group, suggesting a potential connection between the ferroptosis pathway and ALDH1A3 overexpression.

It was found that disulfiram with copper treatment inhibits bladder tumour growth through ferroptosis-mediated ROS induction, which further activates the process of autophagy. Our results prove that DSF-Cu can be an effective add-on therapy along with the standard chemotherapy drugs for the treatment of UBC.

The lesson from many studies investigating the efficacy of targeted therapy in glioblastoma (GBM) showed that a future perspective should be focused on combining multiple target treatments. Our research aimed to assess the efficacy of drug combinations against glioblastoma stem cells (GSCs). Patient-derived cells U3042, U3009, and U3039 were obtained from the Human Glioblastoma Cell Culture resource. Additionally, the study was conducted on a GBM commercial U251 cell line. Gene expression analysis related to receptor tyrosine kinases (RTKs), stem cell markers and genes associated with significant molecular targets was performed, and selected proteins encoded by these genes were assessed using the immunofluorescence and flow cytometry methods. The cytotoxicity studies were preceded by analyzing the expression of specific proteins that serve as targets for selected drugs. The cytotoxicity study using the MTS assay was conducted to evaluate the effects of selected drugs/candidates in monotherapy and combinations. The most cytotoxic compounds for U3042 cells were Disulfiram combined with Copper gluconate (DSF/Cu), Dacomitinib, and Foretinib with IC₅₀ values of 52.37 nM, 4.38 µM, and 4.54 µM after 24 h incubation, respectively. Interactions were assessed using SynergyFinder Plus software. The analysis enabled the identification of the most effective drug combinations against patient-derived GSCs. Our findings indicate that the most promising drug combinations are Dacomitinib and Foretinib, Dacomitinib and DSF/Cu, and Foretinib and AZD3759. Since most tested combinations have not been previously examined against glioblastoma stem-like cells, these results can shed new light on designing the therapeutic approach to target the GSC population.