Article
Resveratrol induces apoptosis in GH3 pituitary adenoma cells of the rat
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Published: | June 4, 2012 |
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Objective: Resveratrol is a phytoestrogen with various antiproliferatic and proapoptotic effects. One of the underlying mechanisms is downregulation of apoptosis inhibitors, such as Survivin. In human pituitary adenoma cells, Survivin expression is increased as compared to healthy pituitary gland tissue. This in vitro study aims to analyze the effect of Resveratrol on GH3 pituitary adenoma cells of the rat.
Methods: GH3 cells were cultivated in F-12K medium containing 2.5% fetal bovine serum, 15% horse serum and 1% penicillin/streptomycin. Ethanol served as solvent for Resveratrol. GH3 cells were incubated in culture medium with Resveratrol concentrations from 20 to 100 μM for 48 to 72 hours. Culture medium and Ethanol served as controls. Cells were counted using a hemocytometer. Apoptosis was quantified using an enzyme-linked immunosorbent assay (ELISA, from Roche), which detects free nucleosomes. Relative expression of Survivin as compared to Actin was measured using quantitative real time polymerase chain reaction (qRT-PCR). Statistics were conducted using the free R software package (from The R Foundation). T served as statistical test, whereas p less than 0.05 was supposed to be statistically significant.
Results: GH3 cell counts significantly decreased with growing concentrations of Resveratrol after 72 hours (70.1, 49.4 and 21.3 per cent of control at 20, 50 and 100 μM respectively; p less than 0.005). ELISA significantly demonstrated apoptosis in 2 passages of GH3 cells treated with 100 μM Resveratrol after 48 hours (mean optical density (OD): 2.05, 1.96) as compared to culture medium (mean OD: 0.19, 0.16) and Ethanol (mean OD: 0.18, 0.17; internal blank control OD: 0.05; internal positive control OD: 3.26; p less than 1*10e-08). In 1 cell passage, qRT-PCR of Survivin detected a relative increase in Survivin expression in the Ethanol control (0.48 versus 0.09) and a relative decrease in Survivin expression after treatment with 100 μM Resveratrol (0.02 versus 0.09) as compared to culture medium. The relative changes in Survivin expression still lack confirmation in the second cell passage.
Conclusions: Resveratrol decreases GH3 cell counts in a dose-dependent manner by inducing apoptosis. Apoptosis in GH3 cells may possibly be mediated by Resveratrol-dependent downregulation of Survivin. Further investigation of the potential effects of Resveratrol on pituitary adenoma cells is warranted.