Tissue factor as an effector of angiogenesis and tumor progression in hematological malignancies.

In the last few years, it has become clear that the processes of tumor angiogenesis, metastasis and invasiveness are highly dependent on components of the blood coagulation cascade. One of the key proteins in coagulation is tissue factor (TF). In addition, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. TF significantly participates in tumor-associated angiogenesis and its expression levels have been correlated with the metastatic potential of many types of hematological malignancies. Signaling pathways initiated by both, tissue factor-activated factor VII (TF-FVIIa) protease activation of protein-activated receptors (PARs), and phosphorylation of the TF-cytoplasmic domain, appear to regulate these tumoral functions. Advances in antiangiogenic therapies and preclinical studies with TF-targeted therapeutics are hopeful in the control of tumor growth and metastasis, but continued studies on the regulation of TF are still needed. In the last few years, the use of approaches of functional genomics and proteomics has allowed the discovery of new proteins involved in the origin of the neoplasia and their participation in the development of the disease. This review attempts to establish a cellular and molecular causal link between cancer coagulopathy, angiogenesis and tumor progression in hematological malignancies.

Biosynthesis of ascorbic acid in kidney bean. L-galactono-gamma-lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane

Hypocotyls of kidney beans (Phaseolus vulgaris L.) accumulated ascorbate after preincubation with a number of possible precursors, mainly L-galactono-gamma-lactone (L-GL) and L-gulono-gamma-lactone. The increase in the intracellular ascorbate concentration was parallel to the high stimulation of the L-GL dehydrogenase (L-GLD) activity measured in vitro using L-GL as a substrate and cytochrome c as an electron acceptor. Cell fractionation using a continuous linear Percoll gradient demonstrated that L-GLD is associated with mitochondria; therefore, pure mitochondria were isolated and subjected to detergent treatment to separate soluble from membrane-linked proteins. L-GLD activity was mainly associated with the detergent phase, suggesting that a membrane-intrinsic protein is responsible for the ascorbic acid biosynthetic activity. Subfractionation of mitochondria demonstrated that L-GLD is located at the inner membrane.

PGE1 protection against apoptosis induced by D-galactosamine is not related to the modulation of intracellular free radical production in primary culture of rat hepatocytes.

D-galactosamine (D-GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE1 treatment reduced necrosis and apoptosis induced by D-GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by D-GalN (0-40mM). Also, the present study investigated if PGE1 (1 microM) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H2O2), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of D-GalN concentrations (2.5-10mM) induced apoptosis in association with a moderate oxidative stress. High D-GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with D-GalN (2.5-10 mM)-treated cells. Although PGE1 reduced apoptosis induced by D-GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.

The calcimimetic R-568 increases vitamin D receptor expression in rat parathyroid glands.

We previously demonstrated that extracellular calcium regulates vitamin D receptor (VDR) expression by parathyroid cells. Since the calcimimetic R-568 potentiates the effects of calcium on the calcium-sensing receptor, it was hypothesized that administration of R-568 may result in increased VDR expression in parathyroid tissue. In vitro studies of the effect of R-568 on VDR mRNA and protein were conducted in cultures of whole rat parathyroid glands and human hyperplastic parathyroid glands. In vivo studies in Wistar rats examined the effect of R-568 and calcitriol alone and in combination. Incubation of rat parathyroid glands in vitro with R-568 (0.001-1 microM) resulted in a dose-dependent decrease in parathyroid hormone (PTH) secretion and an increase in VDR expression (mean +/- SE). Incubation in 1 mM calcium + 0.001 microM R-568 elicited an increase in VDR mRNA (306 +/- 46%) similar to the maximum increase detected with 1.5 mM calcium (330 +/- 42%). In vivo, VDR mRNA was increased after administration of R-568 (168 +/- 9%, P < 0.001 vs. control) or calcitriol (198 +/- 16%, P < 0.001 vs. control). Treatment with R-568 also increased VDR protein in normal rat parathyroid glands and in human parathyroid glands with diffuse, but not nodular, hyperplasia. In conclusion, the present study shows that the calcimimetic R-568 exerts a stimulatory effect on VDR expression in the parathyroid glands of study models and provides additional evidence for the use of calcimimetics in the treatment of secondary hyperparathyroidism.

Cyclin D3 expression in primary Ta/T1 bladder cancer.

Cyclin D3 deregulation has recently been reported in bladder cancer but its prognostic significance remains uncertain. A cohort of 159 patients with stage Ta or T1 primary bladder tumours was investigated to determine the significance of cyclin D3 expression in association with other G1-S phase regulators of the cell cycle (p53, p21Waf1, p27kip1, cyclin D1), including tumour proliferation (ki67-MIB1); its association with conventional clinicopathological parameters; and the relationship between cyclin D3 and loss of heterozygosity (LOH) at the 9p21 (p16INK4a locus) chromosome region. The end point of the study was progression-free survival. Cyclin D3, other G1-S phase regulators, and tumour proliferation were investigated by immunohistochemistry and measured by the grid-counting method. To validate the immunohistochemical expression, cyclin D3 was additionally assessed by western blotting in selected cases. LOH at the 9p21 chromosome region (marker D9S171) was assessed in 125 cases using an AB Prism 310 genetic analyser and a set of microsatellite fluorescence-labelled primers. Cyclin D3 overexpression was related to larger tumour size (>5 cm; p < 0.0001) and high tumour proliferation (>10%; p = 0.025). Mean cyclin D3 expression increased with 2004 WHO grading categories in stage Ta (p = 0.035, ANOVA) and stage T1 (p = 0.047, t test) tumours. Cyclin D3 was not related to other clinicopathological parameters, G1-S phase modulators, or 9p21 LOH. Cox's multivariate analysis selected cyclin D3 as an independent predictor of progression-free survival (p = 0.0012, relative risk (RR) = 5.2366) together with tumour size (p = 0.0115, RR = 4.4442) and cyclin D1 (p = 0.0065, RR = 3.3023). Cyclin D3 expression had the highest risk ratio. Our results suggest that expression of cyclin D3 is relevant to the progression-free survival of patients with Ta/T1 bladder carcinomas.

TNF-alpha dependent production of inducible nitric oxide is involved in PGE(1) protection against acute liver injury.

BACKGROUND: Tumour necrosis factor alpha (TNF-alpha) and nitric oxide modulate damage in several experimental models of liver injury. We have previously shown that protection against D-galactosamine (D-GalN) induced liver injury by prostaglandin E(1) (PGE(1)) was accompanied by an increase in TNF-alpha and nitrite/nitrate in serum. AIMS: The aim of the present study was to evaluate the role of TNF-alpha and nitric oxide during protection by PGE(1) of liver damage induced by D-GalN. METHODS: Liver injury was induced in male Wistar rats by intraperitoneal injection of 1 g/kg of D-GalN. PGE(1) was administered 30 minutes before D-GalN. Inducible nitric oxide synthase (iNOS) was inhibited by methylisothiourea (MT), and TNF-alpha concentration in serum was lowered by administration of anti-TNF-alpha antibodies. Liver injury was evaluated by alanine aminotransferase activity in serum, and histological examination and DNA fragmentation in liver. TNF-alpha and nitrite/nitrate concentrations were determined in serum. Expression of TNF-alpha and iNOS was also assessed in liver sections. RESULTS: PGE(1) decreased liver injury and increased TNF-alpha and nitrite/nitrate concentrations in serum of rats treated with D-GalN. PGE(1) protection was related to enhanced expression of TNF-alpha and iNOS in hepatocytes. Administration of anti-TNF-alpha antibodies or MT blocked the protection by PGE(1) of liver injury induced by D-GalN. CONCLUSIONS: This study suggests that prior administration of PGE(1) to D-GalN treated animals enhanced expression of TNF-alpha and iNOS in hepatocytes, and that this was causally related to protection by PGE(1) against D-GalN induced liver injury.