Evidence of TGF-ß1 mediated epithelial-mesenchymal transition in immortalized benign prostatic hyperplasia cells.

Expression of epithelial-mesenchymal transition (EMT) markers has been detected clinically in benign prostatic hyperplasia (BPH) tissues. To understand the molecular basis, we investigated the role of stromal microenvironment in the progression of EMT in BPH cells. First, we used cell culture supernatant from normal prostate stromal WPMY-1 cells to provide supernatant-conditioned medium (WSCM) to culture the BPH-1 cell line. Then, the morphological changes and migratory capacity were detected in BPH-1 cells. The expression of EMT markers was examined in BPH-1 cells by Western blot and immunofluorescent analysis. Finally, to investigate the role of transforming growth factor beta 1 (TGF-ß1) in this process, the WSCM-cultured cells were treated with monoclonal antibody against TGF-ß1 to study its effect on EMT. We found that the morphology of BPH-1 cells changed to a spindle-like shape after cultured in WSCM, and the levels of E-cadherin and cytokeratin 5/8 (CK5/8) were significantly lower than the cells cultured in ordinary medium. These BPH-1 cells were also tested positive for mesenchymal markers vimentin and a-smooth muscle actin (SMA) as well as Snail. We also found WSCM can increase the migratory capacity of BPH-1 cells. In addition, when they were treated with anti-TGF-ß1, upregulation of E-cadherin and CK5/8 levels was observed but no expression of vimentin, alpha-SMA or Snail was detected. Furthermore, phosphorylated-Smad3 expression in WSCM-cultured BPH-1 cells was also suppressed by anti-TGF-ß1 treatment. Our results demonstrated that stromal cell supernatant was able to induce EMT in BPH-1 cells, possibly through secreting TGF-ß1 to activate Smad signaling. Our results suggest novel molecular targets for clinical treatment of BPH by modification of stromal microenvironment through inhibiting TGF-ß1/Smad expression.

[Effects of clinically effective dose of lovastatin on prostate cancer PC3 cells].

OBJECTIVE: To investigate the effects of clinically achievable dose of lovastatin on prostate cancer PC3 cells. METHODS: PC3 prostate cancer cells were treated with dimethyl sulfoxide(DMSO),or lovastain only,or lovastatin with mevalonic acid for 24, 48 and 72 hours respectively. MTT assay was used to detect the cell viability. By means of [3H] thymidine incorporation tests, the effects of lovastatin on cell proliferation were analyzed. Western blot was used to detect activated casepase3, caspase7, and cleaved PARP (cPARP), the important molecules on the apoptosis pathway. RESULTS: Cell proliferation of PC3 was significantly inhibited by 39.29%[(63.69%+/-3.69%) vs (102.98%+/-6.84%), P=0.000] after 48 h treatment with lovastatin at its clinically achievable dose of 2 micromol/L. After 72 hours the cell proliferation was inhibited by 44.24% [(52.79%+/-9.88% ) vs (97.03%+/-0.87%), P=0.048]. The cell number was also markedly decreased (4.86x10(5)+/-0.10x10(5)) vs (9.66x10(5)+/-0.10x10(5)), P=0.000] after 72 h treatment at this low concentration of 2 micromol/L. The viability of PC3 cells was significantly decreased 50.12% (56.52%+/-6.40%) vs (106.64%+/-5.27%), P=0.000] and 60.05% (41.99%+/-11.64%) vs (102.94%+/-8.49%), P=0.000] after 48 h and 72 h treatment, respectively. In addition, 2 micromol/L lovastatin induced activation of casepase7 and led the death substrate PARP to cleavage. CONCLUSION: Clinically achievable dose of lovastatin inhibits prostate cancer PC3 cell proliferation and induces PC3 cell apoptosis.

[Toxin-neutralizing monoclonal antibodies to the different domains of anthrax protective antigen].

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). PA binds to target cells and transports LF or EF into the cell cytosol where they carry out their enzymatic functions. PA can induce protective immunity to the infection of the bacterium and is the major component in the only anthrax vaccine approved by FDA of USA. Mouse hybridoma clones specifically secreting anti-PA monoclonal antibodies (MAbs) were generated by cell fusion technique and their ability to neutralize anthrax lethal toxin activities was screened in vitro on a toxin-sensitive cell line. Nine toxin-neutralizing MAbs obtained were then characterized for the domains of PA they recognize, and the epitope regions they bind were analyzed by competitive binding ELISA. It was found that these MAbs bind four potential neutralizing epitope regions in three different domains of PA. Four MAbs bind to two non-overlapping epitope regions in domain 4 of PA and may prevent the binding of PA to its cell receptor. Four MAbs bind to domain 2, a domain involved in membrane insertion. One MAb binds to domain 3, a region involved in the oligomerization of PA. The results provided supporting evidence that PA has several neutralizing epitopes, and offered potential immunotherapeutic agents for the treatment of anthrax.