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  • To identify inhibitors of the Hh GLI signaling

    2021-10-19

    To identify inhibitors of the Hh/GLI signaling pathway from natural resources, we have recently reported the successful construction of a cell-based screening assay system for the Hh/GLI signaling pathway (Fig. 1). This is an assay using a GLI-dependent luciferase reporter in human keratinocyte cells (HaCaT) expressing GLI1 under tetracycline control (T-REx system). The 12 consecutive GLI-binding sites (12×GACCACCCA) and the TK promoter were inserted into the pGL4.20 plasmid (Promega). The constructed plasmid, pGL4-GLI BS, was stably transfected into HaCaT cells expressing exogenous GLI1 protein under tetracycline control. During the screening of natural resource libraries, including plant extracts and actinomycete extracts with the assay system, we identified some natural products and natural plant extracts as GLI1-mediated transcriptional inhibitor samples. Among them, we chose the methanol extract of Zizyphus cambodiana (Rhamnaceae) to examine active components, and isolated some GLI-mediated transcriptional inhibitors. Finally, we determined the effect of these inhibitors on protein expression related to the Hh/GLI signaling pathway.
    Results The methanol extract of Z. cambodiana was partitioned between H2O and n-hexane, ethyl acetate. Both n-hexane and ethyl acetate-soluble fractions showed GLI1-mediated transcriptional inhibitory activity at 100μg/mL, and revealed the presence of nearly the same constituents by TLC analysis, so both extracts were combined. The combined extracts was subjected to silica gel, ODS and/or Sephadex LH-20 column chromatography and further purification by reversed-phase HPLC under the guidance of GLI1-mediated transcriptional inhibitory activity. We isolated three active compounds (1, 2 and 3), together with two analogues (4 and 5), which have no inhibition activity. These isolated compounds were identified as colubrinic UNC669 synthesis (1),18, 19 betulinic acid (2), alphitolic acid (3),21, 22 ceanothanolic acid (4), and ceanothic acid (5), on the basis of comparisons with their spectral data in the literature (Fig. 2). All of the compounds have the same structure framework, a pentacyclic triterpene, with difference at the A-ring. Colubrinic acid (1), betulinic acid (2), and alphitolic acid (3) dose-dependently inhibited GLI1-mediated transcriptional activity with little effect on cell viability (Fig. 3), and 4 and 5 were inactive even at 100μM. The IC50 values of 1, 2 and 3 against GLI1-mediated transcriptional inhibitory activity were 38, 32, 42μM, respectively (Table 1). We further examined the effects of 1 and 2 on the protein expression related to Hh/GLI-mediated transcription (Fig. 4). First, we confirmed the expression of exogenous GLI1 protein in HaCaT cells under tetracycline control by Western blotting, and sufficient GLI1 protein was expressed even after treatment with each concentration of 1 at almost the same level (Fig. 4A). Because PTCH and BCL2 expressions are known to depend on GLI-mediated transcription, we investigated the levels of PTCH and BCL2 by Western blotting in GLI1-overexpressing HaCaT cells. Compound 1 showed a dose-dependent reduction of the levels of PTCH and BCL2 proteins. Compound 2 decreased BCL2, PTCH and GLI1 proteins. Because GLI1 was regulated by GLI2, which was the protein affected by Hh signaling, some different mechanism from compound 1 might be included in the case of HaCaT cells. Furthermore, we confirmed the effect of 1 and 2 on the protein expression of PTCH and BCL2 in a human pancreatic cancer cell line (PANC1) (Fig. 4B). The Hh/GLI signaling pathway regulates these protein expressions. Compounds 1 and 2 clearly decreased the expression of PTCH and apoptosis-inhibiting protein BCL2. PANC1 and DU145 express numerous Hh/GLI signaling pathway components, including sonic hedgehog, PTCH, Suppressor of Fused [Su(Fu)], GLI1, and GLI2, resulting from aberrant Hh/GLI signaling in the cells. Next, we confirmed the cytotoxicity of compounds 1–5 against PANC1 and DU145 using a fluorometric microculture cytotoxicity assay (FMCA) (Fig. 5 and Table 1). Compounds 1, 2 and 3 were cytotoxic against PANC1 cells with IC50 values of 43, 44 and 41μM, respectively. Compounds 1, 2 and 3 also showed cytotoxicity against DU145 cells with IC50 values of 78, 37 and 70μM, respectively (Table 1). Compounds 4 and 5 were not Hh/GLI signaling inhibitors, supporting the lower cytotoxicity against PANC1 and DU145. A mesenchymal progenitor (C3H10T1/2) cell line derived from the mouse embryonic mesodermal was also examined, because this normal cell is Hh responsive but not reliant on Hh for survival. Compounds 1, 2 and 3 showed cytotoxicity only at higher concentrations against C3H10T1/2 than those against PANC1 and DU145 cells (Table 1). As shown in Figure 5, it was obvious that normal cells (C3H10T1/2) were less affected by 1 or 2. From the clinical point of view, these results are important because these compounds act as anticancer agents without affecting normal cells.