The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. To search for Hh/GLI inhibitors, we screened for naturally occurring inhibitors of the transcriptional activator GLI1 by using a cell-based assay. We identified zerumbone (1), zerumbone epoxide (2), staurosporinone (9), 6-hydroxystaurosporinone (10), arcyriaflavin C (11) and 5,6-dihydroxyarcyriaflavin A (12) as inhibitors of GLI-mediated transcription. In addition, we isolated physalins F (17) and B (18) from Physalis minima, which are also potent inhibitors. These compounds also inhibited GLI2-mediated transactivation. Semiquantitative RT-PCR and Western blotting analysis further revealed that 1, 9, 17, and 18 decreased Hh-related component expressions. We also show that inhibitors of GLI-mediated transactivation reduce the level of the antiapoptosis Bcl2 expression. Finally, these identified compounds were cytotoxic to PANC1 pancreatic cancer cells, which express Hh/GLI components. These results strongly suggest that the cytotoxicity of the compounds to PANC1 cells correlates with their inhibition of GLI-mediated transcription.

Overexpression of glioma-associated oncogene 1 (GLI1), which has been characterized as a terminal effector and a target gene of the Hedgehog (Hh) signaling pathway, is associated with the development of cancer. A cellular screen was applied utilizing of a GLI-dependent luciferase reporter in human keratinocyte cells (HaCaT) and identified two terpenoids (1 and 2) and a flavonoid glycoside (5) from Acacia pennata as Hh/GLI inhibitors. Compounds 1, 2, and 5 exhibited selective cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells with no toxic effect on normal cells. This result was consistent with a dose-dependent reduction of the protein levels of antiapoptotic BCL-2 and the tumor suppressor patched 1 protein (PTCH). Additionally, treatment of 1 downregulated mRNA expression of Ptch in PANC1, suggesting that the compound has an inhibitory effect on the transcription of Hh/GLI.

Aberrant Wnt/beta-catenin signaling has recently been implicated in tumorigenesis. On the basis of our screening program targeting inhibition of TCF/beta-catenin transcriptional activity, a plant extract of Eleutherine palmifolia was selected as a hit sample. Activity-guided fractionations led to the isolation of 15 naphthalene derivatives (1-15), including 4 new glucosides, eleutherinosides B-E (1-4), and 10 of the 15 compounds showed strong activities with high viability among 293T cells. Our data showed that 2 and 9 inhibited the transcription of TCF/beta-catenin in SW480 colon cancer cells in a dose-dependent manner. These two compounds also showed selective cytotoxicity against three colorectal cancer cell lines. In addition, treatment with 9 led to a significant decrease in the level of nuclear beta-catenin protein, suggesting this reduction to have resulted in the inhibitory effect of 9 on the transcription of TCF/beta-catenin.

Three new natural onoceranoid triterpenes, lansionic acid (1), 3beta-hydroxyonocera-8(26),14-dien-21-one (2), and 21alpha-hydroxyonocera-8(26),14-dien-3-one (3), were isolated from the fruit peel of Lansium domesticum together with two known triterpenoids (4 and 5), and their structures were elucidated from spectral data. These triterpenoids exhibited mild toxicity against brine shrimp (Artemia salina).