Evaluation of a bioluminescent mouse model expressing aromatase PII-promoter-controlled luciferase as a tool for the study of endocrine disrupting chemicals.

Dysfunction of the enzyme aromatase (CYP19) is associated with endocrine pathologies such as osteoporosis, impaired fertility and development of hormone-dependent cancers. Certain endocrine disrupting chemicals affect aromatase expression and activity in vitro, but little is known about their ability to do so in vivo. We evaluated a bioluminescent mouse model (LPTA®)CD-1-Tg(Cyp19-luc)-Xen) expressing luciferase under control of the gonadal aromatase pII promoter as an in vivo screening tool for chemicals that may affect aromatase expression. We studied the effects of forskolin, pregnant mare serum gonadotropin and atrazine in this model (atrazine was previously shown to induced pII-promoter-driven aromatase expression in H295R human adrenocortical carcinoma cells). About 2-4 out of every group of 10 male or female Cyp19-luc mice injected i.p. with 10 mg/kg forskolin had increased gonadal bioluminescence after 3-5 days compared to controls; the others appeared non-responsive. Similarly, about 4 per group of 9 individual females injected with pregnant mare serum gonadotropin had increased ovarian bioluminescence after 24 h. There was a statistically significant correlation between ovarian bioluminescence and plasma estradiol concentrations (n=14; p=0.022). Males exposed to a single dose of 100 mg/kg or males and females exposed to 5 daily injections of 30 mg/kg atrazine showed no change in gonadal bioluminescence over a 7 day period, but a significant interaction was found between atrazine (100 mg/kg) and time in female mice (p<0.05; two-way ANOVA). Ex vivo luciferase activity in dissected organs was increased by forskolin in testis, epididymis and ovaries. Atrazine (30 mg/kg/day) increased (30%) luciferase activity significantly in epididymis only. In conclusion, certain individual Cyp19-luc mice are highly responsive to aromatase inducers, suggesting this model, with further optimization, may have potential as an in vivo screening tool for environmental contaminants.

Antiandrogenic mechanisms of pesticides in human LNCaP prostate and H295R adrenocortical carcinoma cells.

Several pesticides suspected or known to have endocrine disrupting effects were screened for pro- or antiandrogenic properties by determining their effects on proliferation, prostatic-specific antigen (PSA) secretion and androgen receptor (AR) expression, and AR phosphorylation in androgen-dependent LNCaP human prostate cancer cells, as well as on the expression and catalytic activity of the enzyme CYP17 in H295R human adrenocortical carcinoma cells, an in vitro model of steroidogenesis. Effects on SRD5A gene expression were determined in both cell lines. Benomyl, vinclozolin, and prochloraz, but not atrazine, concentration dependently (1-30 µM) decreased dihydrotestosterone (DHT)-stimulated proliferation of LNCaP cells. All pesticides except atrazine decreased DHT-stimulated PSA secretion, AR nuclear accumulation, and AR phosphorylation on serines 81 and 213 in LNCaP cells. Benomyl and prochloraz, but not vinclozolin or atrazine, decreased levels of CYP17 gene and protein expression, as well as catalytic activity in H295R cells. In the case of prochloraz, some of these effects corresponded with cytotoxicity. H295R cells expressed AR protein and SRD5A1, but not SRD5A2 transcripts. SRD5A1 gene expression in H295R cells was increased by 10 nM DHT, whereas in LNCaP cells significant induction was observed by 0.1 nM DHT. AR protein expression in H295R cells was not increased by DHT. Vinclozolin decreased DHT-induced SRD5A1 gene expression in LNCaP, but not H295R cells, indicating a functional difference of AR between the cell lines. In conclusion, pesticides may exert antiandrogenic effects through several mechanisms that are cell type-specific, including AR antagonism and down-regulation or catalytic inhibition of androgen biosynthetic enzymes, such as CYP17 and SRD5A1.

Proliferative and androgenic effects of indirubin derivatives in LNCaP human prostate cancer cells at sub-apoptotic concentrations.

Certain indirubin derivatives are potent cyclin-dependent kinase (CDK) and glycogen synthase kinase (GSK-3ß) inhibitors and may be effective against various cancers. We evaluated the effects of aloisine A, alsterpaullone, aminopurvalanol, indirubin-3'-oxime, 6-Br-indirubin-3'-oxime, kenpaullone, olomoucine and roscovitine on cell proliferation, prostate-specific antigen (PSA) expression, androgen receptor (AR) activation, and GSK-3ß and ß-catenin expression in androgen-dependent LNCaP human prostate cancer cells. Effects were also evaluated in MDA-kb2 human breast cancer cells containing an AR-responsive luciferase construct. Steroid-deprived LNCaP cells were exposed to indirubins±dihydrotestosterone (DHT, 0.1 nM) and cell proliferation was assessed by MTT assay after 120 h. PSA expression was determined by real-time quantitative RT-PCR after 24h. Cytoplasmic and nuclear GSK-3ß/ß-catenin expression and phosphorylation status was determined by Western blotting. Effects on MDA-kb2 luciferase expression were determined after 24h using Steady-Glo (Promega). Indirubin-3'-oxime, 6-Br-indirubin-3'-oxime, alsterpaullone and kenpaullone increased LNCaP cell proliferation and PSA expression (0.03-1 µM; apoptosis occurred >1 µM), whereas aminopurvalanol significantly (p<0.05) reduced DHT-stimulated PSA expression (31%) at 1 nM. The other indirubin derivatives had no effect. The same was observed for induction of AR-dependent MDA-kb2 luciferase expression. Kenpaullone (1, 3 µM) decreased the active- and increased the inactive form of cytoplasmic GSK-3ß, and increased nuclear AR and ß-catenin accumulation. Flutamide (10 µM), unexpectedly, also strongly increased nuclear ß-catenin accumulation. Indirubin derivatives that were potent GSK-3ß inhibitors (relative to CDK1) stimulated LNCaP cell proliferation and other androgenic responses, suggesting (in a cancer treatment context) these compounds may increase AR-dependent prostate cancer growth if not used within an appropriate therapeutic dose-range.