Abstract. Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Adhesion receptors of the ß1 integrin family are assumed to be involved in carcinogenesis, but it is not clear how they contribute to RCC progression. In an in vitro model, we evaluated growth and adhesion capacity of Caki-I and KTC-26 kidney carcinoma cell lines compared to normal renal proximal tubular epithelial cells (PTC). ·1-·6ß1 integrin subunits in malignant and non-malignant cells were evaluated by Western blotting and RT-PCR, integrin surface expression was measured by flow cytometry and confocal microscopy. Additionally, tumor cells were allowed to re-differentiate in the presence of valproic acid (VPA) and dynamic alterations of the integrin profile were analyzed. Caki-I and KTC-26 were characterized by accelerated proliferation and adhesion to an endothelial cell monolayer, compared to PTC cells. The integrin ß1 repertoire in RCC cell lines was significantly different from that detected in PTC, and included down-regulated ·2 and ·6, but up-regulated ·1, ·3 and ·5 proteins. VPA application reduced tumor malignancy which was evidenced by reduced cell growth and adhesion capacity. The reduction in tumor malignancy was paralleled by the integrin expression profile of renal tumor cells 'matching' the pattern seen in PTC. We assume that a sensitive integrin balance exists in normal renal epithelial cells, and that dysregulation of the 'physiological' receptor equipment drives these cells towards malignancy. VPA acted on all investigated integrin subtypes and restored the receptor pattern typical for non-malignant cells. Therefore, VPA may represent a novel therapeutic option in RCC treatment.
Hepatocyte growth factor (HGF) accelerates tissue regeneration and ameliorates tissue fibrosis through its ligand c-Met receptor tyrosine kinase. Hence, HGF is currently discussed as an attractive therapeutic candidate for fatal liver diseases. However, it remains unclear whether c-Met of dedifferentiated hepatocytes adequately responds to HGF in an impaired liver. Therefore, we investigated c-Met expression and c-Met responsiveness to HGF in an experimental dedifferentiation cell culture system. Primary rat hepatocytes were seeded on a two-dimensional collagen matrix or embedded within a three dimensional collagen gel to guarantee intact cell geometry. Cells were cultivated in a growth factor enriched extracellular milieu (de-differentiation medium), or in a chemically defined differentiation medium, representing physiologically intact hepatocytes. c-Met surface expression was determined by flow cytometry. Receptor localisation was examined by confocal microscopy, c-Met and phosphorylated c-Met protein were determined by Western blotting. Hepatocyte-specific asialoglycoprotein receptor (ASGPr) was examined to control the differentiation status of the cells. Growth factor enriched milieu induced a rapid loss of ASGPr with a significant increase of c-Met surface level and a decrease in c-Met protein level. Surprisingly, the increased amount of c-Met surface expression was associated with its loss of responsiveness to HGF. The addition of bile acids into the culture medium had significantly delayed the process of de-differentiation and restrained the drastic elevation of c-Met (tauroursodeoxycholic acid > ursodeoxycholic acid). Application of the three-dimensional hepatocellular architecture stabilized the c-Met surface receptor level and rendered c-Met activation. We have demonstrated that growth factor enriched extracellular milieu and loss of intact liver architecture seems to be accompanied by an up-regulation of c-Met surface level. Our findings suggest that irresponsiveness of c-Met to soluble HGF was possibly caused by an excessive HGF production and receptor over-stimulation. Both events should be considered when establishing an HGF-based therapy for fibrosis/cirrhosis.
CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.
Immune-suppressive agents such as methylprednisolone and cyclosporine exert tremendous side effects, because of high dosage and long-term application required for immune suppression after organ transplantation. Major side effects of methylprednisolone include bleeding of the gastro-intestinal tract, hypertension, and osteoporosis, whereas cyclosporine is nephrotoxic. Liposomes are phospholipid particles that allow delivery of drugs preferentially to the reticuloendothelial system. They can be prepared from phospholipids such as lecithin from soybean or egg yolk, other specific or modified lipids or from membrane-spanning tetraether lipid (TEL), which can be extracted and purified from archaeal cell membranes. One advantage in the use of liposomal application is reduced toxicity of many drugs. We report on various liposomal preparations of cyclosporine, methylprednisolone (L-MPL) and its palmitate derivative (L-MPLP). It has been documented that liposomal cyclosporine A (L-CsA), 1.75 mg/kg/day for seven days has potential for use as an immune-suppressive agent in rats with increased efficacy and decreased nephrotoxicity compared to commercially available forms of intravenous CsA. Liposomal methylprednisolone (L-MPL) 2 mg/kg, intravenously (IV), twice a week shows significantly prolonged cardiac allograft survival in rats and tissue-selective sequestration of the drug in comparison with the same dosage regimen of methylprednisolone in solution, administered daily. We report on organ distribution of L-MPLP in rats after intraperitoneal (IP) administration. Conclusion: Liposomal preparations of immunosuppressants have significantly higher immune-suppressive potential and lower toxicity than non-liposomal preparations. Bipolar TEL can be extracted, fractionated and purified from archaea to form stable liposomes which are extremely resistant, even to gastric fluid. Hence, TEL liposomes allow (besides IV and IP) for oral administration of immuno suppressants after organ transplantation with pharmacological and toxicological advantages as common liposomal phospholipid bilayer preparations.
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