Humans ; Ependymoma/pathology* ; Nuclear Receptor Coactivator 1/metabolism*

OBJECTIVETo investigate the inhibition of the expression of steroid receptor coactivator-1 (SRC-1) in the LNCap cell line through RNA interference (RNAi) and the effect of the silenced SRC-1 gene on LNCap cells.

METHODSThe experiment included four groups: siRNA transfection, siRNA negative control, bland vehicle (with Lipofectamine 2000 but no siRNA), and blank control (with neither Lipofectamine 2000 nor siRNA). LNCap cells were transfected with designed siRNA using the liposomes method, the expressions of SRC-1 determined by Q-PCR and Western blot, and the proliferation of the LNCap cells detected by the CCK-8 method.

RESULTSThe expression of SRC-1 mRNA in the transfected LNCap cells was decreased by 35% at 24 hours and 77% at 48 hours, with statistically significant differences from the blank control group (P < 0.05). The SRC-1 protein expression of the transfected group was 0.359 +/- 0.034 at 24 hours and 0.257 +/- 0.065 at 48 hours, markedly decreased as compared with that of the negative control (0.782 +/- 0.078 and 0.766 +/- 0.043) , bland vehicle (0.840 +/- 0.013 and 0.786 +/- 0.051), and blank control group (0.816 +/- 0.065 and 0.805 +/- 0.107) (P < 0.05). The LNCap cell growth inhibition rates were 25%, 52%, 55% and 60% at 24, 48, 72 and 96 hours, respectively.

CONCLUSIONThe expression of SRC-1 is correlated with the growth of LNCap cells; its high expression in androgen-independent prostate cancer cells may be involved in the progression to androgen-independence. Inhibiting the expression of SRC-1 may be an option for the treatment of androgen-dependent prostate cancer.

Cell Line, Tumor ; Gene Silencing ; Humans ; Male ; Nuclear Receptor Coactivator 1 ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics

The aim of this study was to obtain the soluble protein of human pregnane X receptor ligand binding domain (PXRLBD) through the coexpression of PXRLBD and 88 amino acids of steroid receptor coactivator-1 (SRC88) and apply the protein to constructing a new model of screening PXR ligands. Expression plasmid of pETDuet-1-SRC88-PXRLBD was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88 via induction by IPTG at low temperature. Then an equilibrium dialysis model was constructed to study the interaction between PXRLBD and drugs including clotrimazole and dexamethasone, using HPLC as the analysis method. The results showed that the soluble protein of PXRLBD was obtained and the HPLC data indicated that clotrimazole bound to PXRLBD, while dexamethasone did not bind to PXRLBD, which indicated the successful establishment of a new method for studying the interaction between PXR and drugs. The new method may be useful in the screening of PXR ligands in vitro.
Clotrimazole ; metabolism ; Dexamethasone ; metabolism ; Dialysis ; methods ; Drug Interactions ; Escherichia coli ; genetics ; metabolism ; Histone Acetyltransferases ; genetics ; metabolism ; Humans ; Ligands ; Nuclear Receptor Coactivator 1 ; Plasmids ; Protein Binding ; Receptors, Steroid ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transformation, Genetic

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in several pathological processes of cardiovascular diseases. In this study, the effects of XCT790, a potent and selective inverse agonist of estrogen-related receptor alpha (ERRalpha), on rat VSMCs proliferation and related signal pathways were investigated. The proliferative activity of VSMCs was determined by CCK-8 assay. The mRNA levels of ERRalpha, PGC-1alpha, OPN and MCAD were assayed by RT-PCR. The protein levels of ERRalpha, ERK2 and p-ERK1/2 were evaluated by Western blotting. ELISA was used to assess the protein expression of VEGF. The results showed that XCT790 (5-20 micromol x L(-1)) inhibited rat VSMCs proliferation, and the expression of ERRalpha and its target genes, as well as p-ERK1/2, were also inhibited. XCT790 inhibited VSMCs proliferation in a dose-dependent manner at the dose range from 5 to 20 micromol x L(-1) and in a time-dependent manner at the dose range from 10 to 20 micromol x L(-1). These findings demonstrate that XCT790 inhibits rat VSMCs proliferation by down-regulating the gene level of ERRalpha and thus inhibiting the ERK signal pathway, suggesting that ERRalpha may be a novel potential target for therapeutic approaches to inhibit VSMCs proliferation, which plays an important role in several cardiovascular diseases.
Animals ; Cadherins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytoskeletal Proteins ; genetics ; metabolism ; Dose-Response Relationship, Drug ; GTPase-Activating Proteins ; genetics ; metabolism ; MAP Kinase Signaling System ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Nitriles ; administration & dosage ; pharmacology ; Nuclear Proteins ; genetics ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Phosphorylation ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; genetics ; metabolism ; Thiazoles ; administration & dosage ; pharmacology ; Transcription Factors ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism