Combination Treatment with NCX 4040 and Napabucasin Triggers Enhanced Oxidative Stress, STAT3 Inhibition and Plausible Immunogenic Activation in Metastatic Prostate Cancer Cells

Prostate cancer (PCa) remains a significant health concern, ranking as the second most common cancer after lung cancer, despite recent advances in diagnostics and therapeutics. Due to its heterogeneous nature, diverse etiology, and the limited efficacy of current treatments, developing a therapeutic approach that can address the multifaceted aspects of the disease is imperative. Combinatorial therapy has emerged as a promising strategy for addressing tumor heterogeneity and the limitations of current treatments across various cancer types. In our study, we investigated the potential of combining NCX 4040, a nitric oxide-releasing aspirin derivative, and Napabucasin, a STAT3 and cancer stemness inhibitor, as a novel PCa treatment strategy. We utilized two cellular models: BPH-Cd, a cadmium-transformed carcinogenic cell line, and DU145, a brain metastatic PCa cell line. Our findings demonstrate that the combination treatment exerted a synergistic and dose-dependent reduction in cell viability, tumorigenicity, and migratory capabilities in both BPH-Cd and DU145 cells, surpassing the effects observed with individual treatments. Furthermore, this combination treatment triggered a robust generation of cellular and mitochondrial reactive oxygen species (ROS), resulting in G2/M cell cycle arrest and late-stage apoptosis in both cell lines. Additionally, the combination treatment downregulated redox-sensitive transcription factors pSTAT3 Tyr705 and Ser727, as well as pro-survival proteins TRX1/2, TRXR1, and GPX4, while upregulating tumor suppressor proteins PRDX1 and TXNIP. This implies that inhibition of the pro-survival redox proteins leads to the accumulation of oxidized PRDX1 and TXNIP, thereby elevating oxidative stress and blocking STAT3-mediated transcription, ultimately impeding cell proliferation and survival. Moreover, increased expression of pH2AX protein, a DNA damage marker, indicated that the combination induced DNA damage, resulting in the activation of the cGAS-STING pathway, an anti-tumor immunogenic mechanism as confirmed with immunoblotting. Downregulation of various stem cell markers linked to Wnt/{beta}-catenin and Notch-1 signaling pathways highlight the combinations ability to also target cancer stemness. In summary, our study underscores the promise of combining NCX 4040 and Napabucasin as an innovative and multifaceted therapeutic approach for PCa. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/675191v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@485f6dorg.highwire.dtl.DTLVardef@170f98forg.highwire.dtl.DTLVardef@1037b8org.highwire.dtl.DTLVardef@ba3f39_HPS_FORMAT_FIGEXP M_FIG C_FIG (A) The combination therapy modulates key markers of the redox signaling pathway, inducing excessive oxidative stress in PCa cells, which subsequently leads to DNA damage and late apoptotic cell death. DNA damage activates the cGAS-STING pathway, upregulating interferon regulatory factor 3 (IRF3), which may, in turn, trigger the activation of tumor-infiltrating lymphocytes, generating a potent anti-tumorigenic response. (B) The combination therapy inhibits stem-cell signaling pathways, including Wnt/{beta}-catenin, Notch-1, and STAT3, potentially suppressing cancer stemness and contributing to long-term disease control.