Abstract 192: Honokiol effectively induces apoptosis by targeting the hedgehog signaling pathways in breast cancer

Breast cancer (BrCa) is a heterogeneous and deadly cancer affecting women, involving various pathways regulating the growth and proliferation of cancer cells. Upon diagnosis, BrCa is typically treated according to the molecular subtypes such as estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). In recent years, chemotherapy and targeted therapy have improved the 5-year survival rate for patients with receptor-positive BrCa. However, the patients diagnosed with triple-negative breast cancer (TNBC) remain unsatisfactory. The existing therapies, such as chemotherapy, cause drug resistance, toxicity, and relapse. Therefore, identifying the alternative approach and molecular targets responsible for BrCa development is crucial for improved therapy. One such target is Sonic Hedgehog (Shh) signaling molecules, which have been linked to the activation of several pathways important for developing BrCa. Shh is a critical component of the Hedgehog (Hh) pathway known for cell differentiation and tissue development. In this study, we investigated the effect of Honokiol (HNK), a dietary photochemical isolated from the Magnolia officinalis tree, on inhibiting the progression of TNBC cells (MDA MB231, MDA MB468) and inducing apoptosis via the Shh signaling pathway. HNK possesses potent anti-angiogenic, anti-microbial, anti-inflammatory, and antitumoral activities against aggressive cancers, including BrCa. However, the effect of HNK in targeting Hh signaling in TNBC cells remains limited. To determine the IC50 values of HNK, an MTT assay was performed for different time intervals with various concentrations (5-100µM). HNK cytotoxicity was estimated using live/ dead cell assay, scratch assay, and clonogenic assays. The expression of Hh signaling molecules and apoptotic markers were quantitated with real-time PCR and western blot assay, and an immunofluorescence assay was performed to observe the Shh expression in both cell lines. Our results indicate that HNK led to lower cell viability at 48 hours of treatment compared to control. The flow cytometric analysis showed that HNK enhanced the apoptosis of TNBC cells. Furthermore, HNK increased the expression of cell cycle inhibitor p21WAF1/CIP1, which induces cell cycle arrest by controlling the downstream molecules, CDK4 and cyclin D1. Additionally, HNK suppresses the expression of NF-kB and Hh molecules (Shh, Gli1, and Ptch1) in both TNBC cells. These findings suggest that HNK may directly affect the Shh signaling pathway by interfering with NF-κB crosstalk activity, thereby preventing the communication between tumor cells and surrounding tissue. The migration assay further supported these results. Overall findings indicate a promising role of HNK in targeting the Shh pathway in TNBC type of breast cancer, which could improve the understanding for further translational research. Citation Format: Santosh K. Singh, Rajesh Singh. Honokiol effectively induces apoptosis by targeting the hedgehog signaling pathways in breast cancer [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 192.