Disulfiram Copper Complex Enhancing the Radiation Sensitivity and Immunogenicity of Esophageal Cancer Cells

Abstract

Radiation therapy (RT) is essential for the effective management of esophageal cancer. However, radioresistance of cancer cells limit its efficiency and drives recurrence, thereby leading to poor outcome for patients. Disulfiram is an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Disulfiram chelates bivalent metals and forms complexes with copper, which enhances its anti-tumor activity. Here, we provide evidence that the disulfiram copper complex enhances radiation sensitivity and the immunogenicity of esophageal squamous cell carcinoma (ESCC) cells. ESCC cells were treated with either disulfiram/copper gluconate, RT, or a combination of the two in order to test the effects on cell viability. Colony formation assay was performed to evaluate the radiation sensitivity of disulfiram copper complex. The expression of immunogenic substances was analyzed by flow cytometry methods and Immunofluorescence techniques. Then apoptosis analysis was also performed. We conclude by assessing the potential molecular mechanisms involved in enhanced antitumor activity of the disulfiram copper complex. Disulfiram copper complex exhibits antitumor activity in ESCC cells. This complex was applied at low concentrations, about one fifth of the IC50 dosage in each cancer cells. The outcomes revealing significant improvement of radiation sensitivity by disulfiram copper complex. Immunogenic cell death (ICD), occurring through the translocation of calreticulin from the cytoplasm to the tumor surface in addition to extracellular release of adenosine triphosphate (ATP) and high mobility group protein 1 (HMGB1), was found to occur upon the combined treatment. In addition, the combined strategies induced tumor cell apoptosis, necrosis, and autophagy. Further analyses revealed that the mechanism responsible for radioresistance was due to the ability of tumor cells to overcome moderate endoplasmic reticulum stress induced by RT alone. However, RT combined with disulfiram copper complex successfully activated endoplasmic reticulum stress signaling pathway, leading to a robust stress-response thereby inducing cancer cell death. In summary, our results indicate that RT acts as an endoplasmic reticulum stress inducer and ICD inducer. The combination of RT with disulfiram copper complex induces a severe stress-response, promotes ICD and enhances the efficacy of RT. Our study demonstrates the potential of drug repurposing, providing novel mechanistic insights, identification of new cancer-relevant targets and encouragement for further analysis.