Disulfiram-Copper Potentiates Anticancer Efficacy of Standard Chemo-therapy Drugs in Bladder Cancer Animal Model through ROS-Autophagy-Ferroptosis Signalling Cascade

Abstract

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

Aims: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN); autophagy; drug repurposing; ferroptosis; reactive oxygen species.; urinary bladder cancer.