Abstract 6791: Evaluation of apoptotic and immune mediators in responsecurrent chemotherapeutic agents using murine breast cancer cell lines: The role of HSP70 and S100A9

Abstract Doxorubicin, Cytoxan and Docetaxel are commonly used chemotherapeutic agents in breast cancer treatment, primarily inducing cell death by different mechanisms. Combining these with novel compounds may enhance efficacy and reduce resistance. 4-HCP, active metabolite of Cytoxan has shown potential as an anticancer agent, and HSP70 and S100A9 are linked to cancer progression, immune response, and apoptosis. JG-231 is a small-molecule inhibitor targeting HSP70, disrupting its chaperone activity and inducing apoptosis, particularly in cancer cells. This study explores the effects of Doxorubicin, Docetaxel, and 4-HCP, combined with JG-231, on apoptosis and immune responses in EMT6 and 4T1 metastatic breast cancer cell lines, focusing on HSP70 and S100A9. Effective doses were determined by MTT assays based on dose-response curves. EMT6 and 4T1 cells were treated with Doxorubicin, Docetaxel, 4-HCP and JG-231, combined with JG-231 for 24 hours, and apoptotic/immune responses were analyzed through gene expression (Caspase-3, Caspase-7, Bax, Bcl-2, myc, Stat-1, TNF, HSP70, S100A9) using RT-PCR. Protein levels were validated by Western Blot, apoptotic cell death and changes in cancer stem cell populations by flow cytometry.Treatment with Doxorubicin, Docetaxel, and 4-HCP, combined with JG-231 significantly induced apoptosis in EMT6 and 4T1 cells. Increased Caspase-3 and Caspase-7 expression and a higher Bax/Bcl-2 ratio confirmed activation of apoptosis. Annexin V-positive cells were higher in treated groups, validating apoptosis induction. Furthermore, the cancer stem cell population was reduced, especially in the 4-HCP-treated group. HSP70 and S100A9 expression levels increased after treatment, highlighting their roles in stress response and immune modulation. These treatments elevated Stat-1, Caspase-3, Caspase-7, TNF, HSPA1A, HSPA1B, HSPA2 and S100A9, demonstrating a broader effect on apoptosis and immune response. While increased levels of Caspase indicate programmed cell death, higher Stat-1 and TNF levels may suggest immune mediated cytoprotection. These effect when combined with the elevated levels of stress-induced HSP1A/B, HSPA2 and inflammatory S100A9 correlates with inflammation and immune modulation. Together our findings may provide some evidence for stress-induced pathways and immune mediated cytoprotection in response commonly used chemotherapeutics in breast cancer and thus combining molecularly targeted therapeutics like JG -231 with standard of care may be more effective. This study was supported by the Scientific and Technological Research Council of TURKEY (TUBİTAK) Project no: 1919B022410990; 1059B192301226. Citation Format: Fatma Gul Ozcan, Hilmi Kaan Alkan, Fulya Koksalar Alkan, Ahmet Burak Caglayan, Sukran Ozdatli Kurtulus, Cordarryl Jones, Hasan Korkaya. Evaluation of apoptotic and immune mediators in responsecurrent chemotherapeutic agents using murine breast cancer cell lines: The role of HSP70 and S100A9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6791.