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Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXR␣ and LXR␤; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXR␣ and LXR␤ in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.

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We have developed a high-content screening (HCS) assay to find activators of Wnt/Frizzled (Wnt/Fzd), a pathway known to be important in bone formation. Utilizing primary human preosteoblasts as a model, activation of the Wnt/Fzd pathway was detected by monitoring the stabilization and translocation of the transcription factor beta-catenin from cytoplasm to the nucleus. Endogenous beta-catenin was detected in preosteoblasts by immunofluorescent staining, and subcellular localization was determined by HCS using the Cellomics (Pittsburgh, PA) ArrayScan IV. Positive controls, including Wnt3A-conditioned medium and inhibitors of glycogen synthase kinase-3beta, resulted in increased nuclear beta-catenin. The assay had a Z'-factor of 0.6 and was conducive to automation for high-throughput screening/HCS. By combining standard immunofluorescence technology with automated fluorescence microscopy, we demonstrate the capability of screening cell-signaling pathways in primary human cells.

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.