Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis.

We evaluated the role of COX-2 pathway in 35 head and neck cancers (HNCs) by analyzing COX-2 expression and prostaglandin E2 (PGE2) production in relation to tumor angiogenesis and lymph node metastasis. COX-2 activity was also correlated to vascular endothelial growth factor (VEGF) mRNA and protein expression. COX-2 mRNA and protein expression was higher in tumor samples than in normal mucosa. PGE2 levels were higher in the tumor front zone in comparison with tumor core and normal mucosa (P<.0001). Specimens from patients with lymph node metastasis exhibited higher COX-2 protein expression (P=.0074), PGE2 levels (P=.0011) and microvessel density (P<.0001) than specimens from patients without metastasis. A significant correlation between COX-2 and tumor vascularization (r(s)=0.450, P=.007) as well as between COX-2 and microvessel density with VEGF expression in tumor tissues was found (r(s)=0.450, P=.007; r(s)=0.620, P=.0001, respectively). The induction of COX-2 mRNA and PGE2 synthesis by EGF and Escherichia coli lipopolysaccharide (LPS) in A-431 and SCC-9 cell lines, resulted in an increase in VEGF mRNA and protein production. Indomethacin and celecoxib reversed the EGF- and LPS-dependent COX-2, VEGF, and PGE2 increases. This study suggests a central role of COX-2 pathway in HNC angiogenesis by modulating VEGF production and indicates that COX-2 inhibitors may be useful in HNC treatment.

Apoptosis : molecular regulation of cell death and hematologic malignancies.

Apoptosis, or programmed cell death, represents in cell biology a functional program as important as cell growth or differentiation. Programmed cell death is of basic importance for the development of multicellular organisms and its basic mechanisms are conserved during the evolution of metazoa. Mammalian cells exhibit several different apoptotic pathways that converge to a common endpoint. Each pathway is triggered by a different stimulus: growth factor default, irradiation, induction of the p53 oncosuppressor protein, glucocorticoid hormones (in lymphocytes), ligand binding to Fas/APO (CD95), or tumor necrosis factor receptor (TNF-R), perforin secreted by cytotoxic T cells (reviewed by Hale et al. [1]). As opposed to necrosis, apoptosis is a "clean" process: as the cell shrinks, the cell membrane turns into the "apoptotic shell," the nucleus is condensed and reduced in volume, and eventually the cell disappears from the tissue, due to phagocytosis by neighboring cells or professional phagocytes, such as macrophages.

Molecular polarity in endothelial cells and tumor-induced angiogenesis.

Endothelial cells expose receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) at the abluminal, basal surface that work as basic regulators of tumor-induced angiogenesis. Their specific localization makes them susceptible to the activity of tumor-released stimulatory factors, like VEGF/VPF, which induce proliferation of the endothelial cell toward the extracellular matrix. At the same time, VEGF/VPF stimulates endothelial cells to expose tissue factor (TF), the high-affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades through the extrinsic pathway, so generating thrombin. Thrombin exerts a number of activities: it forms an extracellular fibrin barrier from the VEGF/VPF-dependent fibrinogen extravasation; it activates progelatinase-A (pro-MMP-2), which destroys the basal membrane, allowing proliferation of endothelial cells (ECs) in the novel tumoral fibrin matrix; finally, it induces EC proliferation, potentiating the VEGF effect. Another important factor exposed at the abluminal endothelial cell surface is membrane type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound metalloproteinase, which also activates progelatinase-A, allowing an alternative pathway to that of thrombin to destroy the basal membrane. In addition, we will see that MT1-MMP is also engaged in a direct, cell-associated fibrinolytic activity, essential for tubulogenesis of the novel outsprouting capillary.

Inhibitory effect of full-length human endostatin on in vitro angiogenesis.

Endostatin, a C-terminal product of collagen XVIII, is a very powerful angiogenesis inhibitor. In vivo experiments in mice indicate that endostatin dramatically reduces tumor mass without causing the onset of any resistance to the treatment. Recently, a 12-aa shorter human endostatin has been purified from plasma, but is ineffective in in vitro angiogenesis assays. Here we report that the full-length human recombinant endostatin has a potent inhibitory activity in in vitro angiogenesis assays. Two powerful angiogenic factors were used to stimulate endothelial cells: FGF-2 and VEGF-165. Endostatin prevented cell growth both in the basal condition and after stimulation with FGF-2 or VEGF-165. Migration of microvascular endothelial cells toward FGF-2 or VEGF-165 was impaired, both when cells were pretreated with the inhibitor and when endostatin was added together with the growth factors. Furthermore, experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostatin was ineffective. This study indicates that human endostatin is a potent angiogenesis inhibitor and suggests its use in human anticancer therapy.

Association between vitamin D receptor gene polymorphism and nephrolithiasis.

AIMS: To study the distribution of vitamin D receptor (VDR) gene alleles in hypercalciuric and nonhypercalciuric nephrolithiasis patients, hypothesizing that distinct biochemical parameters would be associated with different VDR genotypes. METHODS: 12 hypercalciuric, 15 normocalciuric nephrolithiasis patients, and 150 healthy subjects were recruited. The individual genetic pattern for VDR was evaluated by DNA extraction followed by polymerase chain reaction amplification of the VDR gene and digestion with the restriction enzyme BsmI. RESULTS: In the hypercalciuric group, Bb patients represented 50% (6/12); bb patients 33% (4/12), and BB cases were 16% (2/12). The VDR frequency distribution was not statistically different in hypercalciuric patients and controls (Bb 72%; bb 16%; BB 12%). In the nonhypercalciuric group, the prevalence of the bb genotype (7/15; 47%) was thrice the percentage of control subjects, while the percentage of BB patients was similar to that of the control group (2/15; 13%). Patients with the bb haplotype exhibited a higher daily urinary calcium excretion. Among hypercalciuric patients, after a calcium-restricted diet, bb patients showed a 39% reduction in daily urinary calcium excretion in comparison with a nonsignificant 13% reduction observed in BB subjects (p = 0.004). CONCLUSIONS: The effects of VDR gene polymorphism on calcium metabolism contribute to the understanding of the pathogenesis of urinary calculi.

Lack of association between body weight, bone mineral density and vitamin D receptor gene polymorphism in normal and osteoporotic women.

In an ethnically homogeneous population of women living in Tuscany, Italy, the relationships between age, body weight, bone mineral density and the vitamin D receptor (VDR) gene polymorphism were studied, with the objective of recognizing patients at risk for osteoporosis. In 275 women bone mineral density was measured by Dual Energy X-rays Absorptiometry (DEXA). In 50 of them the individual genetic pattern for VDR was evaluated by DNA extraction followed by PCR amplification of the VDR gene, and digestion with the restriction enzyme BsmI. Age and bone mineral density were inversely related (R2 = 0.298). Body weight was associated with bone mineral density (R2 = 0.059), but not with age. In osteoporotic women, mean (+/- SD) body weight was 59.9 +/- 6.5 Kg, lower than that recorded in non osteoporotic women (64.2 +/- 9.4 Kg), even though not significantly different (p = 0.18). No association was found between VDR gene polymorphism, bone density or body weight. The performance of anthropometric and genetic components appear to be poor, and, at least for the time being, bone mineral density measurement by means of MOC-DEXA represents the optimal method to detect women at risk for postmenopausal osteoporosis.

Role of p53 mutations in the radiosensitivity status of tumor cells.

Wild-type p53 is involved in cellular response to DNA damage including cell cycle control, DNA repair and activation of apoptosis. Accumulation of p53 protein following DNA damage may initiate the apoptotic process, resulting in cell death. DNA damage induced by radiation is an example of apoptotic stimulus involving p53. Regulation of apoptosis by p53 can occur through transcriptional regulation of pro-apoptotic (e.g. bax) and anti-apoptotic (e.g. bel-2) factors. Although wild-type p53 usually sensitizes cells to radiation therapy, p53 mutations have a variable effect on radiation response. For example p53 mutations in bone or breast tumors have been found to be associated with resistance to chemotherapeutic drugs or ionizing radiation. Mutated p53 has has been reported to increase sensitivity to radiation and drugs in colorectal and bladder tumors. The present brief commentary tries to find an explanation at molecular level of these conflicting results.

Cox-2, iNOS and p53 as play-makers of tumor angiogenesis (review).

Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids. Nitric oxide synthase (NOS) is the enzyme that catalyzes the formation of nitric oxide (NO), a regulator of vascular permeability, from the guanidino nitrogen atom of L-arginine. Two isoforms of both enzymes occur: a constitutive one, Cox-1 and the inducible counterpart Cox-2; also NOS has a constitutive counterparts (cNOS) and an inducible form, called iNOS. The inducible isoforms of both enzymes are of maximum interest. It has been recently shown that cyclooxygenase-2 (Cox-2) is inducible by a variety of stimuli and that eicosanoids, mainly of the PGE2 species, are inducers of basic regulator of angiogenesis, including VEGF/VPF, bFGF, TGF-beta, PDGF, and endothelin-1. In addition, iNOS is inducible by Cox-2. p53 down-regulates the angiogenic process at various levels: it induces thrombospondin-1, a powerful antiangiogenic factor, down-regulates VEGF and NOS and, in addition, down-regulates hypoxia-induced angiogenesis, either inducing apoptosis or enhancing antiangiogenetic factors. It is noteworthy how important the p53 oncosuppressor is in the angiogenesis of solid tumor growth. Cox-2, iNOS and p53 are thus fundamental play-makers of the angiogenic process: they are discussed in detail and a tentative hierarchical cascade is proposed.

Chronic treatment of human fibroblasts cultures with diacylglycerol induces down-regulation of p53 functional activity.

We found that many spontaneous human tumors exhibit increased levels of endocellular diacylglycerol (DAG) which is synthesized de novo as a byproduct of glycolysis. It has been shown that DAG mimics phorbol esters as a full tumor promoter in mouse skin carcinogenesis. A short term DAG treatment activates protein kinase C (PKC), while a long term "chronic" treatment down-regulates PKC. We show here that chronic treatment of human fibroblast with DAG induces p53 down-regulation and inhibition of p53 functional activity, and protection from UV-induced apoptosis. As PKC phosphorylation is necessary for p53 functional activity, we propose that chronic DAG treatment mimics the same event occurring in vivo for the effect of glycolysis in tumor progression.

Acetylation and phosphorylation of the carboxy-terminal domain of p53: regulative significance.

Three fundamental domains are conventionally distinguished on the p53 molecule: an NH2 domain involved in transcription, a central core domain involved in specific DNA binding to the consensus sequences, and a carboxy-terminal domain of about 30 amino acids working as a basic regulatory domain, exhibiting aspecific DNA binding, tetramerization, and nuclearization. The presence of an unmodified carboxy-terminus does not allow the specific transactivation transcriptional function of the p53 protein. Therefore, for the activation of the protein function the carboxy-terminus must be modified. In the present commentary we discuss the role of two covalent modification events occurring at the carboxy-terminus, namely phosphorylation and acetylation, as well as the specific role of these events in the functional regulation of p53 molecule.

Redundant down-regulation pathways for p53.

The aim of this review is to underline the redundancy of down-regulation pathways for p53, at the light of the two more important degradative systems: calpains and ubiquitin-dependent pathways. The MDM2 feed-back loop is also illustrated, as well as the phosphorylative/dephosphorylative regulation of the latent and active p53 isoforms. The mechanisms prolonging p53 half life, following irradiation, are also discussed.

Complex interplay among apoptosis factors: RB, p53, E2F, TGF-beta, cell cycle inhibitors and the bcl2 gene family.

Apoptosis is a fundamental cell program as important as growth, differentiation and quiescence. It regulates tissue development, homeostasis and it is a basic defence against cancer. The cell can undertake multiple apoptotic pathways, where different elements are involved. In this report, we would like to stress particularly that the p53/RB pathway and its complex, interplay with the bcl2 gene family, where paramount elements of apoptosis regulation are operating. It is generally believed that bcl2 blocks apoptosis at the level of the activation of ICE-(Interleukin Converting Enzymes)-like proteases [1,2]. The interconnection between apoptosis and cell cycle is very important and complex and we will start the story from this very up-to-date point of view.

PI3K signal and DNA repair: a short commentary.

PI3K was originally discovered as a lipid kinase involved in the phosphorylation of the inositol ring in position -3, leading to the synthesis of phosphatidyl-inositol-3-4 bisphosphate. The enzyme purified from rat liver is an heterodimer of two subunits of 85 and 110 KD respectively: it phosphorylates the D3 hydroxyl of phosphoinositides to produce phosphatidyl-inositol-3-phosphate. So far the function of the 3-phospho-inositide is unclear. It is likely that the entire phospholipid serves as a second messenger, since no phospholipase C has yet been found that can cleave the inositol group with a 3 phosphate residue. However the activation targets of this second messenger are still poorly known. Recently a novel/serine/theronine kinase was insolated by three groups and called differently RAC, PKB and AKT. It exhibits sequence homology with protein kinase A and C at the carboxyl terminal, whereas the aminoterminal domain has a plectrin homology. Activation of ATK is inhibited by wortmannin, a specific inhibitor of PI3K at very low concentrations. Furthermore inositol-3-phosphate can activate ATK in vitro. In addition very recently, a linkage of G-protein coupled receptors to the MAP kinase signalled pattern through PI3K has been discovered. But what is downstream of this pathway? 70S6 kinase is an attractive candidate since this kinase, involved in protein synthesis, is activated by AKT in vivo. Interestingly AKT is the cellular protooncogene of v-ATK and this implies that ATK induces a pathway of oncogenic transformation. AKT is inhibited by dominant negative mutants of ras and thus involved in the ras-raf-MAP kinase pathway. The role of PI3K is still indefinite but it must have a paramount importance in cell signalling since nearly all growth factor receptors recruit this enzyme and that the activity of fundamental growth factor receptors like PDGF, EGF and insulin are blocked by the specific inhibitor wortmannin, leading to the conclusion that the PI3K signal is much important in mitogenesis, protein synthesis, membrane ruffling, cell transformation and cell cycle progression.