Hedgehog signaling inhibitors isolated from myxomycetes and a Solanaceae plant

The hedgehog (Hh) signaling pathway has been implicated not only in a variety of developmental processes in wide range of organisms, but also in the formation of various tumors such as pancreas and lung cancers. Targeting Hh signaling, therefore, has been expected as an effective cancer therapeutic strategy.

To find the specific inhibitors of Hh/GLI signaling pathway from natural resources, a cell-based screening assay system targeting transcriptional activator GLI1, which constitutes the final step in the Hh signaling pathway, was constructed. A pGL4-Luc reporter vector inserted with 12×GLI binding sites (12GGACCACCCA) was stably transfected into HaCaT cell line expressing GLI1 under tetracycline repressor control. A suitable protocol for the screening was then established. By using this assay system, we identified staurosporinone, 6-hydroxy-staurosporinone arcyriaflavin C, and 5,6-dihydroxyarcyriaflavin A from myxomycetes; zerumbone and zerumbone epoxide from Zingiber spectabile as inhibitors of GLI1-mediated transcriptional activity. In addition, we isolated physalins F and B from Physalis minima (Solanaceae), which are also potent inhibitors (IC₅₀ values, 0.66 and 0.62µM, respectively). These compounds also inhibited GLI2-mediated transactivation. Semiquantitative RT-PCR and Western blotting analysis revealed that these compounds decreased Hh-related component expressions such as GLI1 and PTCH, and also reduced the level of the antiapoptosis Bcl2 expression. In addition, physalins F and B were cytotoxic against PANC1 pancreatic cancer cells (IC₅₀ values, 2.7 and 5.3µM, respectively), which express Hh/GLI components. These results strongly suggested that the cytotoxicity against PANC1 cells may be correlated with their inhibition of GLI-mediated transcriptional activity [1].

Acknowledgements: We are very grateful to Drs. F. Aberger and G. Regl (University of Salzburg) for tetracycline-regulated HaCaT cells and Dr. R. ToftgErd (Karolinska Institute) for the 12GLIRE-TKO luciferase plasmid, Y. Yamamoto for myxomycetes materials and Dr. T. Koyano and Prof. T. Kowithayakorn for plant materials.

Reference: 1. Hosoya, T. et al. ChemBioChem in press.