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Estradiol (E2) increases not only the cell growth but also the cancer stem cell ( CSC ) proportion in estrogen receptor ( ER )‐positive breast cancer cells. It has been suggested that the non‐canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of  CSC  proportion in  ER ‐positive breast cancer cells. We studied anti‐ CSC  activity of a non‐canonical Hh inhibitor  GANT 61 in  ER ‐positive breast cancer cells. Effects of  GANT 61 on the cell growth, cell cycle progression, apoptosis and  CSC  proportion were investigated in four  ER ‐positive breast cancer cell lines.  CSC  proportion was measured using either the mammosphere assay or  CD 44/ CD 24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of  GANT 61 with antiestrogens on the anti‐cell growth and anti‐ CSC  activities were investigated. E2 significantly increased the cell growth and  CSC  proportion in all  ER ‐positive cell lines. E2 increased the expression levels of glioma‐associated oncogene ( GLI ) 1 and/or  GLI 2.  GANT 61 decreased the cell growth in association with a G1‐S cell cycle retardation and increased apoptosis.  GANT 61 decreased the E2‐induced  CSC  proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2‐induced cell growth and  CSC  proportion. Combined treatments of  GANT 61 with antiestrogens additively enhanced anti‐cell growth and/or anti‐ CSC  activities in some  ER ‐positive cell lines. In conclusion, the non‐canonical Hh inhibitor  GANT 61 inhibited not only the cell growth but also the  CSC  proportion increased by E2 in  ER ‐positive breast cancer cells.  GANT 61 enhanced anti‐cell growth and/or anti‐ CSC  activities of antiestrogens in  ER ‐positive cell lines.

Anti‐cancer stem cell activity of a hedgehog inhibitor GANT 61 in estrogen receptor‐positive breast cancer cells