Fancd2os Reduces Testosterone Production by Inhibiting Steroidogenic Enzymes and Promoting Cellular Apoptosis in Murine Testicular Leydig Cells.

BACKGRUOUND: It is well-established that serum testosterone in men decreases with age, yet the underlying mechanism of this change remains elusive. METHODS: The expression patterns of Fancd2 opposite-strand (Fancd2os) in BALB/c male mice and testicular tissue derived cell lines (GC-1, GC-2, TM3, and TM4) were assessed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. The Fancd2os-overexpressing or knockdown TM3 cells were constructed by infecting them with lentivirus particles and were used to evaluated the function of Fancd2os. The testosterone production was measured using enzyme linked immunosorbent assay (ELISA) and the steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were analysed using RT-PCR. The apoptosis of TM3 cells induced by ultraviolet light or testicular tissues was detected using flow cytometry, Western blot or dUTP-biotin nick end labeling (TUNEL) assays. Pearson correlation analysis was used to assess the correlation between the Fancd2os expression and TUNEL-positive staining in mouse testicular Leydig cells. RESULTS: The Fancd2os protein was predominantly expressed in mouse testicular Leydig cells and its expression increased with age. Fancd2os overexpression inhibited testosterone levels in TM3 Leydig cells, whereas knockdown of Fancd2os elevated testosterone production. Fancd2os overexpression downregulated the levels of StAR, P450scc and 3ß-HSD, while Fancd2os knockdown reversed this effect. Fancd2os overexpression promoted ultraviolet light-induced apoptosis of TM3 cells. In contrast, Fancd2os knockdown restrained apoptosis in TM3 cells. In vivo assays revealed that higher Fancd2os levels and mouse age were associated with increased apoptosis in Leydig cells and decreased serum testosterone levels. Pearson correlation analysis exhibited a strong positive correlation between the expression of Fancd2os and TUNEL-positive staining in mouse testicular Leydig cells. CONCLUSION: Our findings suggest that Fancd2os regulates testosterone synthesis via both steroidogenic enzymes and the apoptotic pathway.

TMPyP4-regulated cell proliferation and apoptosis through the Wnt/ß-catenin signaling pathway in SW480 cells.

BACKGROUND: The aim of this study was to investigate the potential effects of the 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of SW480 cells and the underlying mechanisms by which TMPyP4 exerted its actions. METHODS: After treated with different doses of TMPyP4, cell viability was determined by MTT method, the apoptosis was observed by flow cytometry (FCM) and the expression of Wnt, GSK-3ß, ß-catenin and cyclinD1 was measured by RT-PCR and Western blot analysis. RESULTS: The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of SW480 cells in a dose-dependent manner. In addition, the downregulation of Wnt, ß-catenin and cyclinD1 expression levels was detected in TMPyP4-treated SW480 cells. However, followed by the block of Wnt signaling pathway using siRNA methods, the effects of TMPyP4 on proliferation and apoptosis of SW480 cells were significantly reduced. CONCLUSION: It indicates that the TMPyP4-inhibited proliferation and -induced apoptosis in SW480 cells was accompanied by the suppression of Wnt/ß-catenin signaling pathway. Therefore, TMPyP4 may represent a potential therapeutic method for the treatment of colon carcinoma.

[Inhibitory effects of esophageal cancer related gene 2 on proliferation of human esophageal cancer cell EC9706].

OBJECTIVE: To investigate the inhibitory role of esophageal cancer related gene 2 (ECRG2) on proliferation of human esophageal cancer cell EC9706. METHODS: Recombinant plasmid pcDNA3.1-ECRG2 with ECRG2 open reading frame was constructed. The cells were transfected with either pcDNA3.1 or pcDNA3.1-ECRG2 using Lipofectamin 2000. The expression of ECRG2 protein was examined by Dot Blot analysis. The effects of ECRG2 on cell proliferation and malignant was analyzed by colony formation assay. The variation of P53 and P21 were detected in EC9706 cells with or without expression of ECRG2. RESULTS: The plasmid of pcDNA3.1-ECRG2 was successfully established. Colony formation activity of EC9706/pcDNA3.1-ECRG2 was 18% while that of the control cell was 55% in six well plate (P < 0.05). The activity of anchorage-independent proliferation of EC9706/pcDNA3.1-ECRG2 was lower than that of EC9706/pcDNA3.1 in soft agar. After transfected with pcDNA3.1-ECRG2, the expression of P53 and P21 were higher than control. CONCLUSION: ECRG2 can reduced the abilities of proliferation and of anchorage-independent proliferation of EC9706 cells which is through p53 pathway possibility.