The effects of the standardized extracts of Ginkgo biloba on steroidogenesis pathways and aromatase activity in H295R human adrenocortical carcinoma cells

OBJECTIVES: Aromatase inhibitors that block estrogen synthesis are a proven first-line hormonal therapy for postmenopausal breast cancer. Although it is known that standardized extract of Ginkgo biloba (EGb761) induces anti-carcinogenic effects like the aromatase inhibitors, the effects of EGb761 on steroidogenesis have not been studied yet. Therefore, the effects of EGb761 on steroidogenesis and aromatase activity was studied using a H295R cell model, which was a good in vitro model to predict effects on human adrenal steroidogenesis. METHODS: Cortisol, aldosterone, testosterone, and 17β-estradiol were evaluated in the H295R cells by competitive enzyme-linked immunospecific assay after exposure to EGb761. Real-time polymerase chain reaction were performed to evaluate effects on critical genes in steroid hormone production, specifically cytochrome P450 (CYP11/17/19/21) and the hydroxysteroid dehydrogenases (3β-HSD2 and 17β-HSD1/4). Finally, aromatase activities were measured with a tritiated water-release assay and by western blotting analysis. RESULTS: H295R cells exposed to EGb761 (10 and 100 μg/mL) showed a significant decrease in 17β-estradiol and testosterone, but no change in aldosterone or cortisol. Genes (CYP19 and 17β-HSD1) related to the estrogen steroidogenesis were significantly decreased by EGb761. EGb761 treatment of H295R cells resulted in a significant decrease of aromatase activity as measured by the direct and indirect assays. The coding sequence/ Exon PII of CYP19 gene transcript and protein level of CYP19 were significantly decreased by EGb761. CONCLUSIONS: These results suggest that EGb761 could regulate steroidogenesis-related genes such as CYP19 and 17β-HSD1, and lead to a decrease in 17β-estradiol and testosterone. The present study provides good information on potential therapeutic effects of EGb761 on estrogen dependent breast cancer.

Interaction of Pituitary Adenylate Cyclase-Activating Polypeptide and Angiotensin II on Aldosterone Production in Human Adrenocortical H295R Cells / 대한내분비학회지

BACKGROUND: Evidence is accumulating that aldosterone secretion can be regulated in a paracrine and/or an autocrine manner by several neuropeptides locally released within the adrenal gland. Among neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) is present in high concentration in the human adrenal gland. The purpose of this study was to investigate the action of PACAP and the interaction between PACAP and angiotensin II (AII), the main physiologic aldosterone secretagogue, in aldosterone production in human H295R adrenocortical cells. METHODS: H295R cells were incubated with increasing concentrations of PACAP (10(-11)M~10(-7)M) in the absence or presence of 10(-7)M AII. Aldosterone concentration in the supernatant was determined by RIA. Intracellular cAMP content was measured by RIA and total inositol phosphate (IP) production by anion exchange chromatography. Gene expression of CYP11B2 was studied by RT-PCR. RESULTS: In H295R cells, PACAP stimulated aldosterone production in a dose-dependent manner. Incubation of H295R cells with PACAP in the presence of AII significantly increased aldosterone production, compared with that of PACAP alone. PACAP dose-dependently increased cAMP production, but 10(-7)M AII had no effect on either basal or PACAP-stimulated cAMP production. Total IP production was not affected by PACAP, but was increased by 10(-7)M AII; an increase that was not further increased by addition of PACAP. RT-PCR analysis of H295R cells which were exposed to 10-7M PACAP or 10(-7)M AII showed an increase in CYP11B2 transcript signal. Induction of CYP11B2 mRNA expression in response to treatment with both PACAP and AII was significantly more than that resulting from using PACAP alone. CONCLUSION: The present study demonstrates that PACAP exerts a direct stimulatory effect on aldosterone production through induction of CYP11B2 mRNA expression by adenylate cyclase activation as the main intracellular signal pathway in H295R cells. Furthermore, there may be some additive effects between PACAP and AII on aldosterone production.