Evaluation of an epithelial plasticity biomarker panel in men with localized prostate cancer.

BACKGROUND: Given the potential importance of epithelial plasticity (EP) to cancer metastasis, we sought to investigate biomarkers related to EP in men with localized prostate cancer (PC) for the association with time to PSA recurrence and other clinical outcomes after surgery. METHODS: Men with localized PC treated with radical prostatectomy at the Durham VA Medical Center and whose prostatectomy tissues were included in a tissue microarray (TMA) linked to long-term outcomes. We performed immunohistochemical studies using validated antibodies against E-cadherin and Ki-67 and mesenchymal biomarkers including N-cadherin, vimentin, SNAIL, ZEB1 and TWIST. Association studies were conducted for each biomarker with baseline clinical/pathologic characteristics an risk of PSA recurrence over time. RESULTS: Two hundred and five men contributed TMA tissue and had long-term follow-up (median 11 years). Forty-three percent had PSA recurrence; three died of PC. The majority had high E-cadherin expression (86%); 14% had low/absent E-cadherin expression. N-cadherin was rarely expressed (<4%) and we were unable to identify an E-to-N-cadherin switch as independently prognostic. No associations with clinical risk group, PSA recurrence or Gleason sum were noted for SNAIL, ZEB1, vimentin or TWIST, despite heterogeneous expression between patients. We observed an association of higher Ki-67 expression with Gleason sum (P=0.043), National Comprehensive Cancer Network risk (P=0.013) and PSA recurrence (hazard ratio 1.07, P=0.016). CONCLUSIONS: The expression of EP biomarkers in this cohort of men with a low risk of PC-specific mortality was not associated with aggressive features or PSA relapse after surgery.

IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct.

Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells

Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.