R. William G. Watson

Fibrogenesis in Crohn's Disease

INTRODUCTION:Over one-third of patients with Crohns disease (CD) will develop an intestinal stricture and the great majority of these will require at least one surgical procedure. While the pathogenesis of inflammation in CD has been extensively investigated, knowledge of stricture pathogenesis remains limited. The aim of this review is to discuss the current understanding of fibrogenesis in CD and to outline potential directions in research and therapeutics.METHODS:The electronic literature (January 1966 to May 2006) on CD-associated fibrosis was reviewed. Further references were obtained by cross-referencing from key articles.RESULTS:CD-associated fibrosis results from chronic transmural inflammation and a complex interplay among intestinal mesenchymal cells, cytokines, and local inflammatory cells. The fibroblast is the key cell type mediating stricture formation. The cytoarchitecure of the bowel wall is altered with disruption of the muscularis mucosa, thickening of the muscularis propria, and deposition of collagen throughout. The cytokine TGF-β appears critical in this process, acting to increase growth factor and extracellular matrix (ECM) production and dysregulate ECM turnover. Potential therapeutic interventions are likely to concentrate on modulating down-stream targets of TGF-β.CONCLUSIONS:Greater understanding of the biology of fibrostenosis is likely to yield significant advances in our ability to care for patients with stricturing CD. Potential dividends of this approach include identification of novel therapeutic targets and biomarkers useful for prognostication and therapeutic monitoring.

Heat shock proteins HSP27, HSP60, HSP70, and HSP90: expression in bladder carcinoma.

Heat shock proteins (HSPs) are synthesized by cells in response to various stress conditions, including carcinogenesis. The expression of HSPs in neoplasia has been implicated in the regulation of apoptosis, and HSPs also can act by increasing immunity. In the current study, the authors attempted to clarify the significance of HSPs in bladder carcinoma and their effect on tumor behavior.

Regulation of Fas antibody induced neutrophil apoptosis is both caspase and mitochondrial dependent

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co‐stimulation by lipopolysaccharide (LPS) or granulocyte macrophage‐colony stimulating factor (GM‐CSF) on these events. Fas engagement by an agonistic anti‐Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM‐CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co‐stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

BackgroundThere is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel.ResultsThe resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel.ConclusionThis study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Prostate epithelial cell differentiation and its relevance to the understanding of prostate cancer therapies

Prostate cancer is the most common malignancy in males in the western world. However, little is known about its origin and development. This review highlights the biology of the normal prostate gland and the differentiation of basal epithelial cells to a secretory phenotype. Alterations in this differentiation process leading to cancer and androgen-independent disease are discussed, as well as a full characterization of prostate epithelial cells. A full understanding of the origin and characteristics of prostate cancer epithelial cells will be important if we are to develop therapeutic strategies to combat the heterogeneous nature of this disease.

The Apoptosome Pathway to Caspase Activation in Primary Human Neutrophils Exhibits Dramatically Reduced Requirements for Cytochrome c

Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the “apoptosome” as a result of the cell stress–associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell–free extracts readily supported Apaf-1–dependent caspase activation, suggesting that these cells may assemble cytochrome c–independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1–dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1–dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.

Transforming Growth Factor-β Promotes Pro-fibrotic Behavior by Serosal Fibroblasts via PKC and ERK1/2 Mitogen Activated Protein Kinase Cell Signaling

Objective:To assess the role of fibroblasts, transforming growth factor (TGF)-β, and cell signal pathways in promoting fibrosis in Crohns disease (CD). Summary Background Data:Intestinal strictures are a major source of morbidity in CD. Fibroblasts found at sites of stricture promote fibrogenesis. The mechanisms underlying this pro-fibrotic behavior remain elusive. Methods:Fibroblasts were isolated from strictured and macroscopically normal serosa in patients with CD and from normal serosa in patients with colorectal cancer. Whole cell connective tissue growth factor (CTGF) and fibronectin expression were determined by Western blot analysis. Fibroblast type I collagen expression was evaluated by real-time PCR, while fibroblast contractile activity was measured using fibroblast populated collagen lattices. Cells were stimulated with TGF-β1 and inhibitors of the protein kinase C (PKC) and ERK 1/2 mitogen activated protein (MAP) kinase cell signaling pathways. Results:Stricture fibroblasts displayed enhanced constitutive expression of fibronectin. TGF-β promoted fibroblast CTGF, fibronectin, and type I collagen expression and enhanced fibroblast contractile activity. Inhibition of PKC reduced basal collagen expression and contractile activity in Crohns fibroblasts and attenuated the effect of TGF-β on fibroblast CTGF, fibronectin, and collagen I expression as well as fibroblast contractility. ERK 1/2 inhibition had a similar effect on TGF-β-induced CTGF and fibronectin expression. Conclusions:TGF-β is a critical pro-fibrotic growth factor in CD, and its effects are mediated via PKC and ERK 1/2 MAP kinase cell signaling. These pathways may represent novel therapeutic targets for patients with CD characterized by recurrent intestinal stricture formation.

Lipoxin A4 and Aspirin-Triggered 15-Epi-Lipoxin A4 Antagonize TNF-α-Stimulated Neutrophil-Enterocyte Interactions In Vitro and Attenuate TNF-α-Induced Chemokine Release and Colonocyte Apoptosis in Human Intestinal Mucosa Ex Vivo

Lipoxins (LXs) are lipoxygenase-derived eicosanoids and putative endogenous braking signals for inflammation in the gastrointestinal tract and other organs. Aspirin triggers the production of 15-epimers during cell-cell interaction in a cytokine-primed milieu, and aspirin-triggered 15-epi-5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA4) may contribute to the bioactivity profile of this prototype nonsteroidal anti-inflammatory drug in vivo. We determined the effect of LXA4, 15-(R/S)-methyl-11,12-dehydro-LXA4 methyl ester (15-(R/S)-methyl-LXA4), and stable analogs of LXA4 on TNF-α-stimulated neutrophil-enterocyte interaction in vitro and TNF-α-stimulated chemokine release, changes in mucosal architecture, and enterocyte apoptosis in cytokine-activated intact human colonic mucosa ex vivo. LXA4, 15-(R/S)-epi-LXA4, and 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) inhibited TNF-α-stimulated neutrophil adherence to epithelial monolayers at nanomolar concentrations. In parallel experiments involving human colonic mucosa ex vivo, LXA4potently attenuated TNF-α-stimulated release of the C-X-C chemokine IL-8, and the C-C chemokines monocyte-chemoattractant protein-1 (MCP-1) and RANTES. Exposure of strips of normal human colonic mucosa to TNF-α induced disruption of mucosa architecture and enhanced colonocyte apoptosis via a caspase-3-independent mechanism. Prior exposure of the mucosa strips to 15-(R/S)-methyl-LXA4 attenuated TNF-α-stimulated colonocyte apoptosis and protected the mucosa against TNF-α-induced mucosal damage. In aggregate, our data demonstrate that lipoxins and aspirin-triggered 15-epi-LXA4 are potent antagonists of TNF-α-mediated neutrophil-enterocyte interactions in vitro, attenuate TNF-α-triggered chemokine release and colonocyte apoptosis, and are protective against TNF-α-induced morphological disruption in human colonic strips ex vivo. Our observations further expand the anti-inflammatory profile of these lipoxygenase-derived eicosanoids and suggest new therapeutic approaches for the treatment of inflammatory bowel disease.

Thiol‐mediated apoptosis in prostate carcinoma cells

Glutathione (GSH) maintains an optimum cellular redox potential. Chemical depletion, physical efflux from the cell, or intracellular redistribution of this thiol antioxidant is associated with the onset of apoptosis. The aim of this study was to determine the effects of a thiol‐depleting agent, diethylmaleate (DEM), on androgen sensitive and insensitive prostate carcinoma cells.