Interleukin 24 (IL-24) is a tumor-suppressing protein currently in clinical trials. We previously demonstrated that IL-24 leads to apoptosis in cancer cells through protein kinase A (PKA) activation in human breast cancer cells. To better understand the mechanism by which IL-24 induces apoptosis, we analyzed the role of glycogen synthase kinase-3 beta (GSK3β), a highly conserved serine/threonine kinase in cancer cells and a downstream target of PKA. Our studies show for the first time that GSK3β is inhibited following IL-24 treatment in human prostate cancer cells. We showed that the inhibition of GSK3β is mediated through PKA activation triggered by IL-24. IL-24 decreases the phosphorylation of glycogen synthase, substantially activating glycogen synthase and decreasing intracellular glucose levels. Notably, the expression of a constitutively active form of GSK3β abolishes the effect of IL-24. These results demonstrate a previously unrecognized role of IL-24 in apoptosis mediated through GSK3β regulation and its possible implications for metabolic stress, mitochondria dysfunction, and apoptosis. Future studies should precisely delineate the most effective combinations of IL-24 as a GSK3β inhibitor with cytotoxic agents for prostate and other cancers. GSK3β inhibition disrupts average glucose utilization in cancer cells, potentially creating metabolic stress that could be exploited therapeutically.

Background/Objectives: Programmed death ligand 1 (PD-L1) is often overexpressed in triple-negative breast cancer (TNBC), where it helps the tumor evade the immune system and promotes tumor growth. Interleukin-24 (IL-24) is recognized for its anti-tumor activity, although its role in immune regulation remains unclear. In this study, we examined the role of IL-24 in regulating PD-L1 and its anti-cancer activity in TNBC cells. Methods: The study used TNBC cell lines treated with IL-24, delivered via a non-replicating adenovirus vector expressing the IL-24 gene. Assays included MTT for cell viability, Annexin V for apoptosis, Western blot for protein analysis, and qRT-PCR for mRNA analysis. Results: We found that the highly aggressive MDA-MB-231 cells had significantly higher PD-L1 levels. We discovered that treatment with IL-24 reduced cell growth, induced apoptosis, and significantly decreased PD-L1 protein levels in MDA-MB-231 cells. Mechanistically, we identified PKR, also known as eukaryotic translation initiation factor 2 alpha kinase 2, as a key mediator of IL-24–induced PD-L1 suppression. Additionally, doxorubicin, a primary chemotherapy drug used to treat triple-negative breast cancer, decreases PD-L1 expression and increases the sensitivity when combined with IL-24. Conclusions: In this study, we show that IL-24 decreases PD-L1 expression in MDA-MB-231 cells through PKR activation, enhances the anti-tumor effects of Doxorubicin, and may enable lower doses that reduce toxicity and further decrease PD-L1 levels. These findings suggest that IL-24 could serve as a valuable target for therapeutic intervention and suggest that it can improve doxorubicin’s effectiveness against aggressive breast cancer.