Circulating sRAGE in the diagnosis of osteolytic bone metastasis.

Despite the clinical importance of metastasis to the skeleton, the diagnostic tools for early detection and monitoring of bone metastasis lack sensitivity and specificity. We evaluated a promising new serum biomarker, the soluble form of the Receptor of Advanced Glycosylated End-products (sRAGE). sRAGE is involved in the Wnt-signaling pathway, and has been reported to reduce the risk of cancer. We investigated the diagnostic potential of sRAGE to improve the detection and monitoring of bone metastasis. We measured sRAGE in the serum of control healthy subjects, patients with primary tumors and patients with bone metastasis. sRAGE was also correlated with the Wnt inhibitors DKK-1 and sclerostin, the bone resorption markers MMP-2, MMP-9 and TRAP5, and the metastatic marker survivin. sRAGE was significantly lower in primary tumor and metastatic patients than in healthy subjects. sRAGE also showed a strong negative correlation with DKK-1, sclerostin, MMP-2, MMP-9, TRAP5b and survivin. These results indicated that sRAGE might play a protective role in bone metastasis progression, and it may diagnostic significance for detecting and monitoring osteolytic metastases.

Inhibitory effect of HGF on invasiveness of aggressive MDA-MB231 breast carcinoma cells, and role of HDACs.

Hepatocyte growth factor (HGF), through Met receptor binding, fulfils numerous functions in invasive tumour growth (survival/proliferation, motility, apoptosis), but epigenetic control of gene expression in this process is poorly understood. In HGF-treated breast cancer cells we studied (a) the chemoinvasion towards CXCL12 (ligand of the chemokine-receptor CXCR4) and (b) the mechanistic basis, that is, the transduction pathways that regulate CXCR4-mediated invasion, and the role played by histone deacetylases (HDACs) after blockade with trichostatin A (TSA). In highly invasive and metastatic MDA-MB231 cells HGF had a dual inhibitory effect, reducing spontaneous migration and specific chemoinvasion towards CXCL12, the latter by decreasing CXCR4 transactivation and protein level. After HGF the levels of phosphorylated (therefore active) c-Src and Akt persistently increased, indicating a role of these signal transducers in the HGF-dependent cellular and molecular effects. c-Src wild-type expression vector (Srcwt) increased active c-Src and mimicked the HGF-dependent inhibition of CXCR4 transactivation. Our findings indicate that HDACs participated in the HGF-inhibitory effects. In fact, blockade of HDACs hindered the HGF- and Srcwt-dependent reductions of CXCR4 transactivation and invasiveness, while inhibition of endogenous c-Src was additive with HGF, further reducing specific chemoinvasion. In conclusion, in MDA-MB231 cells HDAC blockade with TSA partly counteracted the HGF-dependent effects through molecular events that included enhancement of the expression of the genes for invasiveness Met and CXCR4 (depending on serum conditions), reduction of endogenous phospho-c-Src/c-Src and phosphoAkt/Akt ratios and triggering of apoptosis. The potential therapeutic use of TSA should take into account the variable aggressiveness of breast carcinoma cells and microenvironment signals such as HGF at the secondary growth site of the tumour. It was interesting that HGF reduced motility and CXCR4 functionality only of MDA-MB231 cells, and not of low-invasive MCF-7 cells, suggesting a mechanism implicated in metastatic cell homing.

Diamine oxidase activity in regenerating rat liver and in 4-dimethylaminoazobenzene-induced and Yoshida AH 130 hepatomas.

Diamine oxidase (EC 1.4.3.6) activity, measured as delta 1-[14C]pyrroline formation from [14C]putrescine, was studied in homogenates of regenerating liver and of 4-dimethylaminoazobenzene-induced by Yoshida AH 130 hepatomas of rat. The addition in the incubation medium of acetaldehyde increased delta 1-pyrroline formation in normal and regenerating liver that contained aldehyde dehydrogenase but not in hepatomas where this enzymatic activity was very low or virtually absent. Acetaldehyde did not modify the activity of a preparation of hog kidney diamine oxidase, while chloral hydrate and disulfiram, respectively, enhanced and depressed the activity of this enzyme. These results suggest that aldehyde-metabolizing enzymes present in homogenate may interfere with the amount of delta 1-pyrroline formation and that the use of acetaldehyde may give better information on tissue diamine oxidase activity. Diamine oxidase activity, which was very low in normal liver, increased rapidly in regenerating liver and reached maximum values between 16 and 48 hr after hepatectomy. A large increase in diamine oxidase activity, as compared to the values of normal liver, was also observed in 4-dimethylaminoazobenzene and Yoshida ascites hepatomas.

Regulation of diamine oxidase expression by beta 2-adrenoceptors in normal and hypertrophic rat kidney.

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the beta 2-adrenergic mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, beta 1, beta 2- or beta 2, but not alpha 1-, alpha 2-, or beta 1-receptor-blocking agents prevented this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (alpha 1, alpha 2, beta 1, beta 2), isoproterenol (beta 1, beta 2) or terbutaline (beta 2) showed that also in normal rat kidney diamine oxidase activity is under the control of catecholamine-beta 2-receptors through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered beta 2-receptors coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration.

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy.

Diamine oxidase (EC 1.4.3.6) activity, measured as [14C]delta1 -pyrroline formation from [14C]putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased delta1 -pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with the yield of delta1 -pyrroline formation and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rates prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. The results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzymes rather than to slowing of its degradation.

Diamine oxidase activity induction in regenerating rat liver.

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol.

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.

Influence of proteasome and redox state on heat shock-induced activation of stress kinases, AP-1 and HSF.

We studied the pattern of activation of stress kinases and of transcription factors activator protein-1 (AP-1) and heat shock factor (HSF) in FAO cells by combining two treatments, i.e. heating (42 degrees C for 1 h) and proteasome inhibition, each known to cause cellular heat shock response. The co-treatment heat shock (HS) and proteasome inhibitor (a peptidyl aldehyde or lactacystin) showed cumulative effects on the intensity and duration of activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) at the end of the HS period and during recovery. Similarly, the thiol-reducing agents N-(2-mercaptoethyl)-1,3-diaminopropane and dithiothreitol strongly activated both JNK and p38 MAPK in cells undergoing HS. AP-1 DNA binding activity in response to proteasome inhibitors was so strong that it shadowed the stimulatory effect of HS in the combined treatment, but lactacystin, which is the most potent and specific proteasome inhibitor, decreased the binding late during recovery from HS. Thiol-reducing agents prevented AP-1 DNA binding induced by HS. The combined HS/proteasome inhibitors or HS/thiol-reducing agents treatments cooperatively activated HSF DNA binding. Expression of collagenase I and hsp 70 mRNAs reflects the different behavior of AP-1 and HSF transcription factors in cells exposed to HS and proteasome inhibition. The data seem to indicate that JNK and p38 MAPK activations are not necessarily coupled to DNA binding of AP-1, which can be either increased or inhibited when these kinases are activated. AP-1 and HSF show opposite patterns of response to HS in the presence of proteasome inhibitors or reducing agents.

Influence of polyamines on DNA binding of heat shock and activator protein 1 transcription factors induced by heat shock.

Polyamine depletion, obtained in FAO cells with specific inhibitors of biosynthetic enzymes, prevents or decreases the accumulation of hsp 70 mRNA following heat shock [Desiderio et al., Hepatology 24 (1996) 150-156]. The present study shows that under conditions of spermidine depletion caused by alpha-difluoromethylornithine, the DNA binding capacity of the transcription factor HSF induced by heat shock undergoes a severe and prompt deactivation. Replenishment of the spermidine pool before heat shock re-establishes the DNA binding activity of HSF and the inducibility of hsp 70 mRNA. Similar to HSF, but with a different time-course, the DNA binding of the transcription factor AP-1 activated by heat shock is also impaired in spermidine-depleted cells and reversed by exogenous spermidine. STAT3 provides an example of a transcription factor slightly activated by heat shock but insensitive to polyamine decrease.

Diamine oxidase activity in a model of multistep hepatocarcinogenesis.

Treatment of rats with a complete hepatocarcinogenic regimen (diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy) produced a prolonged stimulation of diamine oxidase activity in liver, which showed early and persistent preneoplastic nodules. Diethylnitrosamine or partial hepatectomy tested separately caused a transient increase in enzyme activity. Early nodules, which were positive for gamma-glutamyltransferase activity, and hepatomas showed diamine oxidase activity of approximately 5- and 13-fold that of control liver, respectively. These results indicate an activation of terminal catabolism of polyamines in preneoplastic nodules and in hepatomas.

Expression patterns of ornithine decarboxylase and c-met in growing Yoshida AH-130 hepatoma.

The expression of the two proto-oncogenes ornithine decarboxylase and c-met was examined during various phases of growth of Yoshida AH-130 ascites hepatoma. Ornithine decarboxylase (ODC) and c-met mRNA levels declined progressively from day 5 (exponential growth-phase) until day 14 (quasi-stationary growth-phase). Transcription rate for both the genes remained constant between days 5 and 10, while decreasing at day 14. ODC activity was consistent with ODC mRNA level during hepatoma growth. In host liver, ODC mRNA accumulated 5 and 14 days after tumor transplantation, while c-met mRNA level was elevated until day 10 and diminished at day 14. ODC activity triplicated at day 14 in host liver. The progressive decline in the expression of ODC and c-met observed in hepatoma might be one of the mechanisms important for the control of tumor growth.

Effect of adrenergic and Ca2+ antagonists on increased ornithine decarboxylase expression in regenerating rat liver.

Partial hepatectomy (PH) (70% resection) causes within 4 hr an accumulation of ornithine decarboxylase (EC 4.1.1.17, ODC) mRNAs concomitant with an increase in ODC activity, maximum values being observed at 8 and 16 hr, respectively. In the early hours of hepatic regeneration, enhancement of transcriptional-rate of ODC gene, demonstrated by nuclear run-on analysis, can account for the accumulation of ODC mRNAs. The involvement of catecholamines in these processes is demonstrated by using prazosin and propranolol, specific antagonists of alpha 1 and beta adrenoceptors, respectively. Prazosin reduces almost completely the rise of ODC activity at 4 hr, without affecting mRNA levels. At 16 hr, enzyme activity and mRNAs increase, however, over the values observed in regenerating liver of prazosin-untreated animals. These findings suggest that alpha 1-receptor activation triggers positive control signals for ODC gene expression at the early time of liver regeneration and, on the contrary, negative signals at later times by mainly post-transcriptional and transcriptional mechanisms, respectively. Propranolol reduces similarly the initial 4 hr-rise of ODC activity. These results indicate that activation of both alpha 1- and beta-adrenoceptors causes the large increase in ODC activity. Pharmacological manipulation of intracellular Ca2+ levels by verapamil, a Ca2(+)-channel blocker, or neomycin, an inhibitor of Ca2+ release from endogenous stores, diminishes ODC activity at 4 and 16 hr after PH. ODC mRNA levels, which are not modified at 4 hr, increase over the values of partially hepatectomized rat liver at 16 hr. Trifluoperazine inhibits both ODC activity and mRNA accumulation at the times studied. As a working hypothesis it is proposed that Ca2(+)-mediated processes induced by catecholamines are involved in ODC gene expression during the prereplicative phase of liver regeneration.

Osteolytic bone metastasis is hampered by impinging on the interplay among autophagy, anoikis and ossification.

Here we show that the fate of osteolytic bone metastasis depends on the balance among autophagy, anoikis resistance and ossification, and that the hepatocyte growth factor (HGF) signaling pathway seems to have an important role in orchestrating bone colonization. These findings are consistent with the pathophysiology of bone metastasis that is influenced by the cross-talk of supportive and neoplastic cells through molecular signaling networks. We adopted the strategy to target metastasis and stroma with the use of adenovirally expressed NK4 (AdNK4) and Dasatinib to block HGF/Met axis and Src activity. In human bone metastatic 1833 cells, HGF conferred anoikis resistance via Akt and Src activities and HIF-1α induction, leading to Bim isoforms degradation. When Src and Met activities were inhibited with Dasatinib, the Bim isoforms accumulated conferring anoikis sensitivity. The proviability effect of HGF, under low-nutrient stress condition, was related to a faster autophagy deactivation with respect to HGF plus Dasatinib. In the 1833 xenograft model, AdNK4 switched metastasis vasculature to blood lacunae, increasing HIF-1α in metastasis. The combination of AdNK4 plus Dasatinib gave the most relevant results for mice survival, and the following molecular and cellular changes were found to be responsible. In bone metastasis, we observed a hypoxic condition - marked by HIF-1α - and an autophagy failure - marked by p62 without Beclin-1. Then, osteolytic bone metastases were largely prevented, because of autophagy failure in metastasis and ossification in bone marrow, with osteocalcin deposition. The abnormal repair process was triggered by the dysfunctional autophagy/anoikis interplay. In conclusion, the concomitant blockade of HGF/Met axis and Src activity seemed to induce HIF-1α in metastasis, whereas the bone marrow hypoxic response was reduced. As a consequence, anoikis resistance might be hampered favoring, instead, autophagy failure and neoformation of woven bone trabeculae. Mice survival was, therefore, prolonged by overcoming an escape strategy adopted by metastatic cells by disruption of tumor-stroma coevolution, showing the importance of autophagy inhibition for the therapy of bone metastasis.

Hepatocyte growth factor in invasive growth of carcinomas.

Hepatocyte growth factor is a multifunctional cytokine of the tumor microenvironment. An important advance in the knowledge of cancer progression has been the appreciation that the tumor invasive phenotype is strongly influenced by microenvironmental stimuli. Malignant tumor cells recruit vasculature and stroma through the production of growth factors and cytokines. The locally activated microenvironment (both cellular and extracellular elements) in turn modifies the proliferative and invasive behavior of the tumor cells. Hepatocyte growth factor accomplishes most of the functions of the invasive program in carcinomas (loss of adhesive junctions, motility, angiogenesis, survival/apoptosis), and may interact with other signals such as hypoxia. The purpose of the present review is to highlight examples of the progress in this area. The influence of hepatocyte growth factors on the carcinoma invasive phenotype is considered by evaluating the gene targets and the network of transcription factors activated in the specific responses.

Hepatocyte growth factor differently influences Met-E-cadherin phosphorylation and downstream signaling pathway in two models of breast cells.

E-cadherins are implicated in cell adhesion, and also in cell signaling by associating with tyrosine kinase-receptors such as Met, the hepatocyte growth factor (HGF) receptor. Using two different cellular models, i.e. MCF-7 (breast carcinoma) and MCF-10 (immortalized mammary) cells, we studied the possible mechanism(s) by which E-cadherins modulate the signaling pathways downstream of Met, leading to beta-catenin-TCF transcriptional activity. In MCF-7, but not in MCF-10 cells, E-cadherins were remarkably associated with Met. Moreover, in MCF-7 cells both co-immunoprecipitation with anti-Met antibody and co-localization were increased by 30-min HGF treatment, which caused E-cadherin tyrosine phosphorylation. Also beta-catenin in the co-immunoprecipitate was phosphorylated by HGF, probably favoring TCF activation. Consistently, after HGF treatment, beta-catenin redistributed earlier in MCF-7 than in MCF-10 cells, with nuclear accumulation and activation of TOPFLASH gene reporter. Our results indicate a functional role of Met-E-cadherin interaction in MCF-7 cells through the amplification of the signaling downstream of HGF-Met triggering that involved c-Src and phosphoinositide-3-kinase activities.

Effect of rate of rise of blood alcohol on outcome of cardiac injury.

In our initial study in anesthetized dogs, single 7-minute intravenous alcohol doses caused a 67% mortality with cardiac impact at 65 mg% blood alcohol. In the next study, equally spaced oral doses over 40 minutes resulted in only a 17% mortality at an equivalent blood alcohol level and injury severity. Intravenous doses of a 50% ethanol/isotonic saline solution were given at equally spaced intervals over 40 minutes to generate peak blood alcohol levels and rates of rise equivalent to the oral study. Impacts with a velocity of 10 m/s and a contact compression of 5 cm were delivered to 85-90% of the right pericardial surface. The 25% mortality from alcohol and trauma was comparable to that observed in the oral study. These results attest to the importance of the rate of alcohol administration over the route on the outcome of cardiac injury.

The potentiation of the response to blunt cardiac trauma by ethanol in dogs.

Acute changes in mechanical performance and electrical activity were followed after blunt cardiac trauma, ethanol infusion, and ethanol infusion and blunt cardiac trauma in 21 anesthetized dogs. Impact was delivered to most of the pericardium with an impact velocity of 10 m/sec and a contact compression of 5 cm. Impact alone caused transient arrhythmias and significant reductions in all hemodynamic parameters with ultimate recovery of rhythm and cardiac performance within 60 minutes after impact. Intravenous infusion of ethanol (average blood alcohol concentration, 65 +/- 1 mg %), resulted in no significant alterations in either mechanical performance or electrical activity but, when combined with trauma, caused a mortality rate of 67%. All animals died from excitation-contraction decoupling; a dissociation of electrical from mechanical activity such that an electrical event does not elicit a mechanical event strong enough to sustain life. It is concluded that even low blood alcohol concentrations can significantly reduce cardiac performance in the presence of otherwise nonfatal cardiac injury.