Claire Morgan

Naturally derived factors and their role in the promotion of angiogenesis for the healing of chronic wounds

Chronic diseases such as vascular disease and diabetes are witnessing a global increase in prevalence. Such diseases often predispose patients to the development of severe, debilitating, chronic wounds. Angiogenesis, the formation of new capillaries from the pre-existing vascular network, is an essential component of wound healing and aberrant angiogenesis is evident in almost all chronic wounds. Natural products, derived from both plants and animals, provide a significant haven of compounds which have proved to be of great benefit to man and his ailments. Whilst significant advances have been made in the understanding of impaired angiogenesis in a non-healing wound, in the clinical setting, few effective agents exist that can expedite wound healing and closure. The lack of effective healing agents has led to a renewed interest in investigations into natural wound healing resources. In this review, we collate new evidence that details the potential for several natural compounds to promote angiogenesis and wound healing, most predominately via the up regulation of VEGF expression, that warrant urgent further investigation for development into new pro-angiogenic/wound healing therapies.

The in vitro anti-tumour activity of zoledronic acid and docetaxel at clinically achievable concentrations in prostate cancer

Bisphosphonates and chemotherapy have increasingly gained favour in the treatment of metastatic hormone resistant prostate cancer. We investigated whether zoledronic acid, at a concentration found at the bone, would enhance the anti-tumour activity of docetaxel in the hormone resistant prostate cancer cell line PC-3. Cells were exposed to zoledronic acid (1 mM) in combination or in sequence with docetaxel (3 nM). Cell viability, apoptosis and markers for inhibition of the mevalonate pathway were analyzed 48 or 72 hours after drug treatment. Reduction in cell viability and increased apoptosis levels were most pronounced with single agent zoledronic acid. Western blot analysis showed an overall reduction in the proliferation marker Mini chromosome maintenance protein 2 (MCM2) and reduction in caspase-3 precursor for all drug treatments and a marked reduction in Rho A levels with single agent zoledronic acid and zoledronic acid-docetaxel sequence. This study highlights the potency of zoledronic acid, when used at concentrations similar to those found at the bone, in reducing cell viability and causing apoptosis. Clinically, these findings suggest that in patients with bone metastases due to hormone resistance prostate cancer, who are not fit enough for systemic chemotherapy, single agent zoledronic acid may have a direct effect on viability of prostate cancer epithelial cells.

In Vitro Acid Exposure has a Differential Effect on Apoptotic and Proliferative Pathways in a Barrett's Adenocarcinoma Cell Line

INTRODUCTION:Acid, a principal component of refluxate, may contribute to the neoplastic progression of Barretts esophagus. Brief acid exposure in vivo and in vitro has been shown to increase cell proliferation. The mechanisms underlying the hyperproliferative response are not well elucidated but may include alterations in Na+–H+ exchanger activity and MAPK signaling pathways.OBJECTIVE:To ascertain the effects of pulsatile acid exposure on gene expression in a Barretts adenocarcinoma cell line (SEG-1).METHODS:SEG-1 cells were exposed to either acidified DMEM at pH 3.5 (0.1 M hydrochloric acid) or pH 7.4 (control) for 20 min followed by neutralization of the medium. Total RNA was extracted before acid exposure and over a 10-h time course (0.5, 2, 4, 6, 8, and 10 hours) and hybridized to Affymetrix human U133A oligonucleotide arrays. Data were analyzed using the Affymetrix statistical expression algorithms. Only alterations in gene expression that were ≥2 and ≤−2 fold were studied further and a subset was further investigated by reverse transcription polymerase chain reaction (RT-PCR) and densitometry. Apoptosis was assayed in SEG-1 cells by western blot for cleaved caspase 3 and an apoptosis ELISA assay.RESULTS:Changes in expression were identified for 138 genes. Analysis of gene function identified immediate downregulation of genes associated with apoptosis and early upregulation of genes associated with proliferation. The gene expression profiles suggest that MAPK pathways may be involved and suppression of apoptosis may occur via p53-dependent mechanisms.CONCLUSIONS:Microarray analysis of gene expression changes in a Barretts adenocarcinoma cell line has identified cellular pathways that may be disrupted following acid exposure.

Putative prognostic epithelial-to-mesenchymal transition biomarkers for aggressive prostate cancer.

Prostate cancer is the second most frequently diagnosed cancer worldwide and is the sixth leading cause of cancer deaths in men, yet it varies greatly in its aggressiveness. Currently, it is not possible to adequately differentiate between patients whose tumors will remain indolent and those patients whose disease will progress, resulting in unnecessary aggressive treatment. Consequently, there is an urgent need to identify markers of prostate cancer progression, invasiveness and metastasis to more accurately predict prognosis. The aim of this study was to assess the ability of key epithelial-to-mesenchymal transition molecules in identifying prostate cancer patients who are likely to develop aggressive tumors. Using 215 archival patient tissue samples, immunohistochemistry was applied to examine the expression and sub-cellular localization of E-Cadherin, Snail, Slug, Twist, Vimentin, BMP-2 and BMP-7. Of the seven markers assessed, a significantly increased expression of Snail protein was observed within the nucleus of prostate cancer cells and was strongly associated with increasing Gleason score and clinical stage. In addition, loss of E-Cadherin expression at the cellular membrane of prostate cancer cells was also significantly associated with increasing Gleason score, clinical stage, and additionally, a reduction in survival.

Quantum Dots for Multiplexed Detection and Characterisation of Prostate Cancer Cells Using a Scanning Near-Field Optical Microscope

In this study scanning near-field optical microscopy (SNOM) has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms and the effect of aberrant adhesion protein expression in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2), E-cadherin was predominantly located around the cell periphery and within filopodial extensions. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. In contrast, examination of metastatic prostate adenocarcinoma cells (PC-3) revealed no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not found to form close cell-cell associations with their neighbours. We have demonstrated that with a fully optimised sample preparation methodology, multiplexed quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.

Increased expression of ARF GTPases in prostate cancer tissue

PurposeARFs are a family of Ras-related GTP binding proteins, ARF6, in particular, is implicated in cancer invasion and metastasis. However, the role of ARF proteins in prostate cancer have yet to be investigated.MethodsImmunohistochemical staining for ARF6 was performed on a prostate cancer tissue microarray with patient matched normal specimens.ResultsAntibody staining was significantly over-expressed in prostate cancer patient samples compared to normal patient tissue and a trend towards increased staining intensity in cancer samples with Gleason scores of 8 and above (metastatic disease).ConclusionDue to high homology between members of the ARF family we could not determine if ARF 6 was the only ARF over-expressed in the prostate cancer samples. However, we are the first to show that ARF-GTPases are over expressed in prostate cancer which provides further insight into the molecular biology of prostate cancer.

A Newly Developed Nested PCR Assay for the Detection of Helicobacter pylori in the Oral Cavity.

Goals: To develop a new nested polymerase chain reaction (PCR) assay for identifying Helicobacter pylori DNA from dental plaque. Background: H. pylori is one of the most common chronic bacterial pathogens in humans. The accurate detection of this organism is essential for proper patient management and for the eradication of the bacteria following treatment. Study: Forty-nine patients (24 males and 25 females; mean age: 51; range, 19 to 94 y) were investigated for the presence of H. pylori in dental plaque by single-step PCR and nested PCR and in the stomach by single-step PCR, nested PCR, and histologic examination. Results: The newly developed nested PCR assay identified H. pylori DNA in gastric biopsies of 18 patients who were histologically classified as H. pylori-positive and 2 additional biopsies of patients who were H. pylori-negative by histologic examination (20/49; 40.8%). Dental plaque samples collected before and after endoscopy from the 49 patients revealed that single-step PCR did not detect H. pylori but nested PCR was able to detect H. pylori DNA in 40.8% (20/49) patients. Nested PCR gave a higher detection rate (40.8%, 20/49) than that of histology (36.7%, 18/49) and single-step PCR. When nested PCR results were compared with histology results there was no significant difference between the 2 methods. Conclusions: Our newly developed nested PCR assay is at least as sensitive as histology and may be useful for H. pylori detection in patients unfit for endoscopic examination.

A role for STEAP2 in prostate cancer progression

Prostate adenocarcinoma is the second most frequent cancer worldwide and is one of the leading causes of male cancer-related deaths. However, it varies greatly in its behaviour, from indolent non-progressive disease to metastatic cancers with high associated mortality. The aim of this study was to identify predictive biomarkers for patients with localised prostate tumours most likely to progress to aggressive disease, to facilitate future tailored clinical treatment and identify novel therapeutic targets. The expression of 602 genes was profiled using oligoarrays, across three prostate cancer cell lines: CA-HPV-10, LNCaP and PC3, qualitatively identifying several potential prognostic biomarkers. Of particular interest was six transmembrane epithelial antigen of the prostate (STEAP) 1 and STEAP 2 which was subsequently analysed further in prostate cancer tissue samples following optimisation of an RNA extraction method from laser captured cells isolated from formalin-fixed paraffin-embedded biopsy samples. Quantitative analysis of STEAP1 and 2 gene expression were statistically significantly associated with the metastatic cell lines DU145 and PC3 as compared to the normal prostate epithelial cell line, PNT2. This expression pattern was also mirrored at the protein level in the cells. Furthermore, STEAP2 up-regulation was observed within a small patient cohort and was associated with those that had locally advanced disease. Subsequent mechanistic studies in the PNT2 cell line demonstrated that an over-expression of STEAP2 resulted in these normal prostate cells gaining an ability to migrate and invade, suggesting that STEAP2 expression may be a crucial molecule in driving the invasive ability of prostate cancer cells.

Does maggot therapy promote wound healing? The clinical and cellular evidence.

The larvae of Lucillia sericata, or maggots of the green‐bottle fly, are used worldwide to help debride chronic, necrotic and infected wounds. Whilst there is abundant clinical and scientific evidence to support the role of maggots for debriding and disinfecting wounds, not so much emphasis has been placed on their role in stimulating wound healing. However, there is accumulating evidence to suggest that maggots and their externalized secretions may also promote wound healing in stubborn, recalcitrant chronic ulcers. There are a growing number of clinical reports which support the observation that wounds which have been exposed to a course of maggot debridement therapy also show earlier healing and closure end‐points. In addition, recent pre‐clinical laboratory studies also indicate that maggot secretions can promote important cellular processes which explain this increased healing activity. Such processes include activation of fibroblast migration, angiogenesis (the formation of new blood vessels from pre‐existing vessels) within the wound bed, and an enhanced production of growth factors within the wound environment. Thus, in this review, we summarize the clinical evidence which links maggots and improved wound healing, and we précis recent scientific studies which examine and identify the role of maggots, particularly individual components of maggot secretions, on specific cellular aspects of wound healing.

The Role of Adhesion Molecules as Biomarkers for the Aggressive Prostate Cancer Phenotype

Background Currently available methods for diagnosis and staging of prostate cancer lack the sensitivity to distinguish between patients with indolent prostate cancer and those requiring radical treatment. Alterations in key adherens (AJ) and tight junction (TJ) components have been hailed as potential biomarkers for prostate cancer progression but the majority of research has been carried out on individual molecules. Objective To elucidate a panel of biomarkers that may help distinguish dormant prostate cancer from aggressive metastatic disease. Methods We analysed the expression of 7 well known AJ and TJ components in cell lines derived from normal prostate epithelial tissue (PNT2), non-invasive (CAHPV-10) and invasive prostate cancer (LNCaP, DU145, PC-3) using gene expression, western blotting and immunofluorescence techniques. Results Claudin 7, α –catenin and β-catenin protein expression were not significantly different between CAHPV-10 cells and PNT2 cells. However, in PC-3 cells, protein levels for claudin 7, α –catenin were significantly down regulated (−1.5 fold, p = <.001) or undetectable respectively. Immunofluoresence showed β-catenin localisation in PC-3 cells to be cytoplasmic as opposed to membraneous. Conclusion These results suggest aberrant Claudin 7, α – and β-catenin expression and/or localisation patterns may be putative markers for distinguishing localised prostate cancer from aggressive metastatic disease when used collectively.