Gregory M. Harrison

Pomegranate (Punica granatum) pure chemicals show possible synergistic inhibition of human PC-3 prostate cancer cell invasion across Matrigel.

SummaryFour pure chemicals, ellagic acid (E), caffeic acid (C), luteolin (L) and punicic acid (P), all important components of the aqueous compartments or oily compartment of pomegranate fruit (Punica granatum), and each belonging to different representative chemical classes and showing known anticancer activities, were tested as potential inhibitors of in vitro invasion of human PC-3 prostate cancer cells in an assay employing Matrigel™ artificial membranes. All compounds significantly inhibited invasion when employed individually. When C, P, and L were equally combined at the same gross dosage (4 μ g/ml) as when the compounds were tested individually, a supradditive inhibition of invasion was observed, measured by the Kruskal-Wallis non-parametric test.

Biphasic effects of 17-β-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells

Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction function are an early and key event during the vascular spread of cancer and inflammation. This study sought to determine the role of estrogen in the regulation of tight junctions and expression of molecules making tight junctions in endothelial cells. Human endothelial cell, HECV, which express ER‐β but not ER‐α was used. 17‐β‐estradiol induced a concentration‐ and time‐dependent biphasic effect on tight junction. At 10−9 and 10−6 M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10−12 M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17‐β‐estradiol at lower concentrations (as low as 10−12 M). Furthermore, the time‐dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 min and the second drop occurring 48 h after 17‐β‐estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin‐1 and ‐5, and ZO‐1, were reduced by 17‐β‐estradiol, in line with changes of TER. This study shows that 17‐β‐estradiol can induce concentration‐ and time‐related biphasic effects on tight junction functions expression of occludin in endothelial cells and that this perturbation of tight junction functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer. J. Cell. Physiol. 196: 362–369, 2003.

Claudin-16 reduces the aggressive behavior of human breast cancer cells

Claudin‐16 (Paracellin‐1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re‐absorption of magnesium and calcium in the kidney. This study examined expression of Claudin‐16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin‐16 levels and patient prognosis. Insertion of the Claudin‐16 gene into MDA‐MB‐231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and “tighter” colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno‐stained. RNA was reverse transcribed and analyzed by Q‐PCR (standardized using β‐actin, normalized with cytokeratin‐19 levels). Levels of expression of Claudin‐16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin‐16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin‐16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin‐16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin‐16 plays a role beyond that of an initial metastasis repressor in this cancer type. J. Cell. Biochem. 105: 41–52, 2008.

The Expression of the Nectin Complex in Human Breast Cancer and the Role of Nectin-3 in the Control of Tight Junctions during Metastasis

Introduction Nectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells. Methods A human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour. Results Q-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation. Conclusions Nectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells.

The Hepatitis A Virus Cellular Receptor, HAVcR-1, Reduces the Integrity of Human Endothelial Tight Junctions.

BACKGROUND Hepatitis A virus cellular receptor-1 (HAVcR-1) is the cellular receptor for Hepatotropic picornavirus. Although HAVcR-1 is expressed in every human organ, its natural function remains unknown. This study investigated the location, association and functionality of HAVcR-1 in human endothelial cells. MATERIALS AND METHODS HAVcR-1 was either overexpressed or knocked-down by plasmid electroporation in human umbilical vein endothelial (HECV) cells. Changes in tight junction (TJ) behaviour were assessed using transendothelial resistance, and paracellular permeability. Binding partners and the location of HAVcR-1 protein was assessed using Western blotting/immunofluorescence. RESULTS HAVcR-1 co-localised with zonula occludens-1 (ZO-1) and ZO-2 proteins, both of which are involved in the formation, maintenance and function of TJ. The overexpression of HAVcR-1 resulted in reduced TJ formation; knockdown cells were resistant to hepatocyte growth factor (HGF)-mediated TJ disruption. HGF was unable to effect reduced resistance in these cells. HAVcR-1 was co-precipitated with the TJ regulatory factor Ras homolog gene family, member C (Rho C). CONCLUSION HAVcR-1 may have a novel function as part of the regulatory apparatus for TJ in human endothelial cells.

β-Catenin, its Binding Partners and Signalling Mechanisms: Implications in Prostate Cancer

β-catenin is a cytoplasmic protein that has been well documented in recent years by a number of investigators. It is now firmly established that β-catenin plays a dual role through its function in facilitating intercellular adhesion by binding to E-cadherin, and also for its key role as a mediator of the Wnt signal transduction pathway. This chapter summarises some of the scientific and clinical advances made during the last decade on the molecular and cellular functions of β-catenin, including its role in Wnt signalling, adherence junction assembly and its impact on prostate cancer progression.