Abstract 2025: Blockade of the Shh pathway by KAAD-Cyclopamine promoted apoptosis in estrogen treated breast cancer cells

Abstract

Abstract The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Recent studies have shown constitutive activation of the hedgehog pathway is important in the development of various carcinomas including breast cancer. However, it remains unclear whether this pathway is activated in human breast cancer. Herein, we investigated the role of Shh signaling pathway in tumorigenesis of estrogen treated breast cancer cells and the molecular mechanisms underlying these effects. We showed that breast cancer cell lines express all the components of Shh signaling, albeit to different extents. Moreover, in breast cancer cells, estrogen supplementation triggered Shh up-regulation. This estrogen treated Shh expression activated the Hh pathway in a ligand-dependent manner, and increased cell proliferation. Cell proliferation was done MTT assay with various concentrations of KAAD-Cyclopamine for 24, 48, or 72 h. As a result, KAAD-Cyclopamine had anti-proliferative effect on estrogen treated breast cancer cells. Also, to determine whether the cytotoxicity induced by KAAD-Cyclopamine was due to apoptosis in estrogen-induced breast cancer cells, we measured cell cycle and the fraction of early apoptosis [Annexin V(+)/PI(-)]. Cell cycle analysis by FACS indicated that Cyclopamine treated cell lines showed an increase in the proportion of cells in sub-G1 phase, compared to untreated cells. Exposure of gastric cancer cells to Cyclopamine also resulted in the increase in the percentage of annexin V- positive and PI-negative cells. These observations demonstrate that KAAD-Cyclopamine can inhibit estrogen-induced breast cancer cell cycle progression and promote cell apoptosis. Furthermore, Western blotting analysis was used to investigate the effect of KAAD-Cyclopamine (a Shh signaling inhibitor) on estroen treated breast cancer cells. As a result, expression of cleaved PARP, caspase-3, and -9, Bax was increased. In addition, phosphorylation of Akt and of glycogensynthase kinase 3β (GSK-3β) was also reduced by treatment with KAAD-Cyclopamine. These results, taken together, suggest that Blockade of the Shh pathway by KAAD-Cyclopamine promoted apoptosis in estrogen treated breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2025. doi:1538-7445.AM2012-2025