Abstract 6400: Synergistic combination of NCX 4040 and napabucasin induces immunogenic activation via upregulation of mitochondrial oxidative stress and STAT3 inhibition in cadmium transformed and metastatic prostate cancer cells
Authors: Pathak Nishtha , Munirathinam Gnanasekar 
Published: April 2023

Abstract
Despite recent diagnostic and therapeutic advances, prostate cancer (PCa) poses a significant burden on men’s health and quality of life, retaining the second highest incidence rate after lung cancer. The combinatorial therapy approach has emerged as a useful treatment option for various types of cancer to target tumor heterogeneity and overcome the limitations of existing treatment modalities. Here, we explored the combination of NCX 4040, a nitric oxide-releasing aspirin derivative and Napabucasin, a recently developed STAT3 and cancer stemness inhibitor as a potential treatment for PCa. Two cellular models were used namely BPH-Cd, a cadmium-transformed carcinogenic cell line and DU145, a brain metastatic PCa cell line. Cell viability assay indicated reduced cell viability in both cell lines in a dose-dependent manner and more so in combination as opposed to individual treatments, with an IC50 of 5 µM for NCX 4040 and 250 nM for Napabucasin. Notably, treatment with catalase prevented the synergistic activity of NCX 4040 and Napabucasin in reducing the viability of these cells. This suggested that NCX 4040 and Napabucasin might be inducing oxidative stress related cellular death. In addition, the combination of NCX 4040 and Napabucasin robustly impeded the in-vitro tumorigenic potential in both cell lines in colony formation assay and decreased the size of spheroids with increasing doses in in-vitro 3D spheroid assay in BPH-Cd. Western blot analysis revealed that in addition to the downregulation of STAT3, NCX 4040 and Napabucasin treatment showed upregulation in cGAS-STING immunogenic activation pathway related markers (pSTING, cGAS, pTBK1 in DU145 and STING, TBK1 in BPH-Cd). This data demonstrated that combination drug treatment might be inducing cell death via immunogenic activation. Additional markers related to this pathway are being tested in both cellular models. Interestingly, thioredoxin (TRX)-interacting protein was highly upregulated in both cell lines following combination treatment indicating that the synergistic effects are mediated via targeting TRX-regulated cellular redox signaling. Subsequently, TRX and thioredoxin reductase 1 were also downregulated in both cell lines. Furthermore, confocal imaging analysis indicated that NCX 4040 and Napabucasin together might be causing DNA damage and mitochondrial stress as DNA release was clearly evident. Flow cytometric analysis demonstrated that the drug combination shows a synergistic effect in producing excess mitochondrial and cellular ROS, causing apoptosis and in inducing cell cycle arrest in both BPH-Cd and DU145 cells. Taken together, our study demonstrates that NCX 4040 and Napabucasin in combination could be a potential anti-PCa therapy which warrants further evaluation for its clinical application.
Citation Format: Nishtha Pathak, Gnanasekar Munirathinam. Synergistic combination of NCX 4040 and napabucasin induces immunogenic activation via upregulation of mitochondrial oxidative stress and STAT3 inhibition in cadmium transformed and metastatic prostate cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6400.