Vaccinium myrtillus L. extract and its native polyphenol-recombined mixture have anti-proliferative and pro-apoptotic effects on human prostate cancer cell lines.

Vaccinium myrtillus berry extract (VME) and a recombined standard mixture (RSM) of its main native phenolic compounds were investigated for cell growth inhibition and pro-apoptotic activity on hormone-dependent (LNCaP) and hormone-independent (PC3 and DU-145) prostate cancer (PCa) cell lines. Normal prostate epithelial cells (PrEC) were also studied in comparison. VME hindered anchorage-dependent PCa cell proliferation in a dose-dependent manner, that is, at 1/800 (v/v) dilution for LNCaP and PC3, and 1/100 (v/v) dilution for DU-145 (corresponding to 14.15 and 113.2 µg cyanidin-3-O-glucoside equivalents per ml of culture medium), respectively. VME had a growth inhibitory effect towards PrEC at the same dilution of DU-145 cells although the IC50 values indicated that PrEC are more resistant than PCa cell lines. VME also reduced the anchorage-independent growth of PCa cells. The study of the apoptotic profile (i.e., non-apoptotic, early apoptotic, late apoptotic and necrotic cells) evidenced that the apoptotic rate (early+late) was statistically higher in all three cell lines exposed to VME compared to control. Anchorage-dependent and anchorage-independent growth inhibition of RSM was very similar to that displayed by VME. Moreover, RSM exerted its growth inhibitory effect also under hypoxia, the latter representing a biological condition known to sustain PCa proliferation and aggressiveness.

Liver dysfunction as predictor of prognosis in patients with amyloidosis: utility of the Model for End-stage Liver disease (MELD) scoring system.

Amyloidosis prognosis is often related to the onset of heart failure and a worsening that is concomitant with kidney-liver dysfunction; thus the Model for End-stage Liver disease (MELD) may be an ideal instrument to summarize renal-liver function. Our aim has been to test the MELD score as a prognostic tool in amyloidosis. We evaluated 128 patients, 46 with TTR-related amyloidosis and 82 with AL amyloidosis. All patients had a complete clinical and echocardiography evaluation; overall biohumoral assessment included troponin I, NT-proBNP, creatinine, total bilirubin and INR ratio. The study population was dichotomized at the 12 cut-off level of MELD scores; those with MELD score >12 had a lower survival compared to controls in the study cohort (40.7 vs 66.3 %; p = 0.006). Either as a continuous and dichotomized variable, MELD shows its independent prognostic value at multivariable analysis (HR = 1.199, 95 % CI 1.082-1.329; HR = 2.707, 95 % CI 1.075-6.817, respectively). MELD shows a lower prognostic sensitivity/specificity ratio than troponin I and NT-proBNP in the whole study population and AL subgroup, while in TTR patients MELD has a higher sensitivity/specificity ratio compared to troponin and NT-proBNP (ROC analysis-AUC: 0.853 vs 0.726 vs 0.659). MELD is able to predict prognosis in amyloidosis. A MELD score >12 selects a subgroup of patients with a higher risk of death. The predictive accuracy seems to be more evident in TTR patients in whom currently no effective scoring systems have been validated.

Carbonic anhydrase IX is a marker of hypoxia and correlates with higher Gleason scores and ISUP grading in prostate cancer.

BACKGROUND: Carbonic anhydrase IX is a member of α-carbonic anhydrases that is preferentially expressed in solid tumors. It enables bicarbonate transport across the plasma membrane, neutralizing intracellular pH and conferring to cancer cells a survival advantage in hypoxic/acidic microenvironments. Overexpression of carbonic anhydrase IX in cancer tissues is regulated by hypoxia inducible factor 1α - mediated transcription and the enzyme is considered a marker of tumor hypoxia and poor outcome. The role of carbonic anhydrase IX in prostate cancer has not been fully clarified and controversy has arisen on whether this enzyme is overexpressed in hypoxic prostate cancer tissues. METHODS: We analyzed the expression of carbonic anhydrase IX and hypoxia inducible factor 1α in two prostate cancer cell lines, LNCaP and PC-3, and in 110 cancer biopsies, by western blotting and immunocyto/histochemistry. RESULTS: In LNCaP and PC-3 cells, carbonic anhydrase IX was mostly cytoplasmic/nuclear, with very limited membrane localization. Nuclear staining became stronger under hypoxia. When we analyzed carbonic anhydrase IX expression in human prostate cancer biopsies, we found that protein staining positively correlated with hypoxia inducible factor 1α and with Gleason pattern and score, as well as with the novel grading system proposed by the International Society of Urological Pathology for prostate cancer. Once more, carbonic anhydrase IX was mainly cytoplasmic in low grade carcinomas, whereas in high grade tumors was strongly expressed in the nucleus of the neoplastic cell. An association between carbonic anhydrase IX expression level and the main clinic-pathological features involved in prostate cancer aggressiveness was identified. CONCLUSIONS: There was a statistically significant association between carbonic anhydrase IX and hypoxia inducible factor 1α in prostate cancer tissues, that identifies the enzyme as a reliable marker of tumor hypoxia. In addition, carbonic anhydrase IX expression positively correlated with prostate cancer grading and staging, and with outcome, suggesting that the protein may be an independent prognosticator for the disease. The nuclear translocation of the enzyme in hypoxic cancer cells may epitomize a biological switch of the tumor towards a less favorable phenotype.

Losartan reduces oxidative damage to renal DNA and conserves plasma antioxidant capacity in diabetic rats.

Increased reactive oxygen species (ROS) levels produced by hyperglycemia and angiotensin-II (AT-II) are considered among the pathogenic factors in the malignant transformation of diabetic renal cells. We aimed to investigate the potential role of AT-II in the increased cancer risk seen in diabetes; measuring oxidative damage to renal DNA and protective antioxidant defenses, including adiponectin (Adp) and plasma antioxidant capacity by the Ferric Reducing Ability of Plasma (FRAP) method. In the kidney of streptozotocin (STZ)-induced (55 mg/kg) diabetic rats either treated or not treated for 3 weeks with losartan, an AT-II type 1 receptor antagonist (20 mg/kg/day); we measured 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels, as an index of oxidative DNA damage, circulating Adp and FRAP. Diabetic rats showed significantly higher 8-oxodGuo levels in renal DNA (8.48 ± 0.98 × 10(-6) dG, mean ± SEM n = 11) than normoglycemic ones (1.18 ± 0.04 × 10(-6) dG, mean ± SEM, n=7) and lower plasma Adp and FRAP levels in comparison to normoglycemics. The treatment of diabetic rats with losartan significantly (P < 0.01) reduced 8-oxodGuo levels (5.4 ± 0.58 × 10(-6) dG, mean ± SEM n=9) in renal DNA and conserved FRAP values. Moreover, an inverse correlation was found between 8-oxodGuo in kidney DNA and circulating Adp levels in normoglycemic and diabetic rats. Losartan treatment preserves FRAP levels, reduces DNA oxidative injury and thus the carcinogenesis risk. Furthermore, our results indicate that Adp plasma levels are a further marker of oxidative injury to the kidney and confirm that it is an important part of the plasma antioxidant defense.

Biohumoral markers as predictor of right ventricular dysfunction in AL Amyloidosis.

AIM: In AL amyloidosis, the importance of right ventricle (RV) involvement has recently been underlined and its role in predicting prognosis has been emphasized. Little is known about the relationship between RV involvement, N-terminal pro-brain natriuretic peptide (NT-proBNP) and troponin levels. Aim of our study was to clarify the relationship between NT-proBNP and troponin and RV involvement and analyze their independent value as predictors of RV dysfunction. METHODS AND RESULTS: We examined 76 consecutive patients with biopsy-proven AL amyloidosis. Each patient received complete clinical evaluation, troponin I, NT-proBNP assay and comprehensive echocardiographic evaluation. Considering a tricuspidal annulus plane systolic excursion (TAPSE) value <16 mm, 23 patients (30%) presented RV systolic dysfunction, whereas 53 (70%) did not. Patient with reduced TAPSE had thicker left ventricle (LV) walls and RV free walls, reduced LV fractional shortening, impaired LV diastolic function and worse LV and RV myocardial performance index. For RV dysfunction the best predictive value for NT-proBNP was identified as 2977 ng/l with sensitivity and specificity of 87% and 84%, respectively; best cut-off for troponin I was identified as 0.085 ng/l, with sensitivity and specificity of 85% and 90% respectively. At multivariable logistic regression analysis, both NT-proBNP and troponin I emerged as independent predictors of RV dysfunction presence but troponin appears to have a higher predictive power. CONCLUSION: Our study demonstrated that cut-off values of 2977 ng/ml for NT-proBNP and 0.085 ng/l for troponin were able to identify a subgroup of AL patients with RV dysfunction. Troponin I is more accurate and seems to be the best biohumoral marker of RV dysfunction.

Transitory FGF treatment results in the long-lasting suppression of the proliferative response to repeated FGF stimulation.

FGF applied as a single growth factor to quiescent mouse fibroblasts induces a round of DNA replication, however continuous stimulation results in arrest in the G1 phase of the next cell cycle. We hypothesized that FGF stimulation induces the establishment of cell memory, which prevents the proliferative response to repeated or continuous FGF application. When a 2-5 days quiescence period was introduced between primary and repeated FGF treatments, fibroblasts failed to efficiently replicate in response to secondary FGF application. The establishment of "FGF memory" during the first FGF stimulation did not require DNA synthesis, but was dependent on the activity of FGF receptors, MEK, p38 MAPK and NFκB signaling, and protein synthesis. While secondary stimulation resulted in strongly decreased replication rate, we did not observe any attenuation of morphological changes, Erk1/2 phosphorylation and cyclin D1 induction. However, secondary FGF stimulation failed to induce the expression of cyclin A, which is critical for the progression from G1 to S phase. Treatment of cells with a broad range histone deacetylase inhibitor during the primary FGF stimulation rescued the proliferative response to the secondary FGF treatment suggesting that the establishment of "FGF memory" may be based on epigenetic changes. We suggest that "FGF memory" can prevent the hyperplastic response to cell damage and inflammation, which are associated with an enhanced FGF production and secretion. "FGF memory" may present a natural obstacle to the efficient application of recombinant FGFs for the treatment of ulcers, ischemias, and wounds.

Skeletal muscle: an endocrine organ.

Tropism and efficiency of skeletal muscle depend on the complex balance between anabolic and catabolic factors. This balance gradually deteriorates with aging, leading to an age-related decline in muscle quantity and quality, called sarcopenia: this condition plays a central role in physical and functional impairment in late life. The knowledge of the mechanisms that induce sarcopenia and the ability to prevent or counteract them, therefore, can greatly contribute to the prevention of disability and probably also mortality in the elderly. It is well known that skeletal muscle is the target of numerous hormones, but only in recent years studies have shown a role of skeletal muscle as a secretory organ of cytokines and other peptides, denominated myokines (IL6, IL8, IL15, Brain-derived neurotrophic factor, and leukaemia inhibitory factor), which have autocrine, paracrine, or endocrine actions and are deeply involved in inflammatory processes. Physical inactivity promotes an unbalance between these substances towards a pro-inflammatory status, thus favoring the vicious circle of sarcopenia, accumulation of fat - especially visceral - and development of cardiovascular diseases, type 2 diabetes mellitus, cancer, dementia and depression, according to what has been called "the diseasome of physical inactivity".

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer.

Prostate cancer (PC) is still the second cause of cancer-related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone-sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non-raft into the raft compartment where it colocalized with the γ-secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.

Resistin level in coronary artery disease and heart failure: the central role of kidney function.

OBJECTIVES: The aim of this study was to evaluate resistin levels in patients with coronary artery disease (CAD) with or without chronic heart failure, in order to define its independent predictor. METHODS: One hundred and seven outpatients with CAD were enrolled in the study and divided into three groups: CAD without left-ventricular systolic dysfunction (group 1); CAD with left-ventricular dysfunction without heart failure symptoms (group 2); CAD with overt heart failure (group 3). Plasma resistin was determined by ELISA. RESULTS: Resistin progressively increased from group 1 (10.7±5.0 ng/ml) to groups 2 (11.8±5.8 ng/ml) and 3 (17.0±6.8 ng/ml), with the difference reaching statistical significance in group 3 versus groups 1 and 2 (P=0.001). A multivariable model of analysis demonstrated that the best predictor of plasma resistin level was the estimated glomerular filtration rate (P<0.001), indicating that reduction of kidney function was the main cause of the adipokine increase observed in patients with CAD and overt heart failure. CONCLUSIONS: Our data confirm the rise of resistin plasma levels previously described in patients affected by chronic heart failure; however, in our study, this relationship seemed to be mediated mainly by the level of kidney function, and only partially by the severity of ventricular dysfunction.

[The complexity of risk stratification in older patient candidate to non-cardiac surgery]. / La complessità della stratificazione del rischio nel paziente anziano cardiopatico candidato a chirurgia non cardiaca.

During the last decades the older patients who are candidates for surgery have grown exponentially due to the increase in life expectancy and the surgery technique improvement. Despite this, the mortality remains high and our ability to predict the surgery outcomes continues to be low in the elderly. The main reason is related to different difficulties; we are unable to differentiate properly the chronological from the biological age, and the current surgery and cardiology risk scores are poorly geriatric-oriented. We must underline how the measure of comorbidity during the preoperative evaluation is often limited to a simple count of comorbid conditions, without a more detailed assessment of their severity. On the other hand different comorbidity scores have been validated in geriatric populations showing a good correlation with prognosis, such as the Index of Coexisting Disease-ICED or the Geriatric Index of Comorbidity-GIC. Our predictive deficiency about the outcomes is linked to poor attention for identifying the frail patients that are already at high risk of disability. Recently, the evaluation of frailty is a key target for geriatric medicine, and geriatricians have developed various methods for measuring this parameter and suggesting the physical performance indexes as a reliable surrogate of frailty. Surrogate frailty measures, such as the "gait speed" or the "Short Physical Performance Battery-SPPB" seem to be the valid tools for evaluating older surgery patients due to their simplicity and short administration time. We think that the future challenge will be their widespread use in this specific clinical setting.

Notch signaling modulates hypoxia-induced neuroendocrine differentiation of human prostate cancer cells.

UNLABELLED: Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. RESULTS: Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and ß3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. CONCLUSIONS: Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription.

Regulation of non-classical FGF1 release and FGF-dependent cell transformation by CBF1-mediated notch signaling.

FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non-classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML. The expression of a dominant negative (dn) form of CBF1 in 3T3 cells induces transcription of FGF1 and sphingosine kinase 1 (SphK1), which is a component of FGF1 export pathway. dnCBF1 expression stimulates the stress-independent release of transduced FGF1 from NIH 3T3 cells and endogenous FGF1 from A375 melanoma cells. NIH 3T3 cells transfected with dnCBF1 form colonies in soft agar and produce rapidly growing highly angiogenic tumors in nude mice. The transformed phenotype of dnCBF1 transfected cells is efficiently blocked by dn forms of FGF receptor 1 and S100A13, which is a component of FGF1 export pathway. FGF1 export and acceleration of cell growth induced by dnCBF1 depend on SphK1. Similar to dnCBF1, dnMAML transfection induces FGF1 expression and release, and accelerates cell proliferation. The latter effect is strongly decreased in FGF1 null cells. We suggest that the regulation of FGF1 expression and release by CBF1-mediated Notch signaling can play an important role in tumor formation.

S100A13 is a new angiogenic marker in human melanoma.

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called 'dysplastic' nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clark's level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7+/-0.7) to vertical growth phase (3.6+/-3.1) to metastases (7.0+/-7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

Multiple shocks affect thoracic electrical impedance during external cardioversion of atrial fibrillation.

BACKGROUND: Thoracic impedance (TI) influences the success of external cardioversion (ECV) or defibrillation because current intensity traversing the heart is inversely related to TI. Experimental data suggest that TI decreases after multiple shocks. We undertook a clinical study to determine changes of TI values in patients with atrial fibrillation or flutter requiring ECV. METHODS: We enrolled 222 consecutive patients (age 73 +/- 11 years; males 67%; body weight 75 +/- 13 kg) who underwent ECV between January 2004 and February 2007. Biphasic shocks were delivered through adhesive pads placed in the anteroposterior position. The initial energy was set at 1 J/kg, with progressive increases up to a maximum of 180 J in case of failure. In the last 39 elective patients, plasma concentration of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha were determined before and 6 hours after ECV. RESULTS: Sinus rhythm was restored in 202 patients (91.0%). Of these, 155 (69.8%) required more than one shock (on average, 2.5 +/- 1.5 shocks/patient). Final values of energy and peak current intensity were 136 +/- 47 J and 50 +/- 14 A, respectively. TI decreased significantly by 6.2% from baseline after > or =2 shocks (P < 0.001). The absolute reduction was correlated with baseline TI, number of delivered shocks, and hemoglobin oxygen saturation. IL-6 and TNF-alpha increased with ECV (P < 0.001 and P = 0.014, respectively). CONCLUSIONS: TI decreases significantly after multiple shocks, possibly by activation of acute inflammation.

Protein folding does not prevent the nonclassical export of FGF1 and S100A13.

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.

The release of fibroblast growth factor-1 from melanoma cells requires copper ions and is mediated by phosphatidylinositol 3-kinase/Akt intracellular signaling pathway.

Melanoma is a highly invasive tumor with elevated mortality rates. Progression and aggressiveness appear related to the achievement of an angiogenic phenotype. Melanoma cells express several angiogenic factors, including fibroblast growth factor (FGF)-1 and FGF-2. The autocrine production and release of FGFs and the subsequent activation of FGF receptors, have a central role in melanoma tumor progression. We demonstrated that FGF-1 is secreted from a human melanoma cell line, A375, under conditions of serum deprivation. The release of FGF-1 is inhibited by the copper chelator ammonium tetrathiomolybdate, suggesting a role of copper in the secretory pathway, and is triggered by activation of phosphatidylinositol 3-kinase (PI3K)/Akt intracellular signaling. Interestingly, overexpression or activation of Akt has been correlated with poor prognosis in melanoma patients. Our data indicate a novel role for Akt in supporting the progression of human melanomas and advocate the need for new treatments targeting PI3K/Akt signaling pathway, to control tumor development and progression.

Photoexposition discriminates Notch 1 expression in human cutaneous squamous cell carcinoma.

The Notch signaling pathway may play opposing roles in cancer. It can be oncosuppressive or protumoral, depending on the cellular and tissue context. In skin cancer, Notch 1 expression is downregulated, thus supporting the hypothesis of an oncosuppressive role in cutaneous carcinomas. However, as members of the Notch family undergo downregulation upon exposure to UV irradiation, we wondered whether Notch 1 expression in skin carcinomas may be governed by additional factors, including UV exposure. We investigated the expression of Notch 1 and its ligands, Jagged 1, Jagged 2 and Delta-like 1, by immunohistochemistry in a series of premalignant and invasive cutaneous carcinomas, including 4 solar keratoses, 5 Bowen's disease, 5 squamous cell carcinomas on sun-exposed skin, 6 squamous cell carcinomas on sun-protected genital skin and 14 basal cell carcinomas of different histotypes (nodular, superficial type, sclerodermiform/infiltrating and baso-squamous). Expression of Notch 1 was decreased in solar keratoses and invasive squamous cell carcinomas localized on sun-exposed skin. In contrast, marked Notch 1 staining was observed in extragenital Bowen's disease as well as in genital (penile) human papilloma virus-related in situ and invasive squamous cell carcinomas. A diffuse Notch 1 staining was detected in nodular and superficial basal cell carcinomas while sclerodermiform/infiltrating and baso-squamous basal cell carcinomas showed a low to absent Notch 1 expression. Jagged 1, Jagged 2 and Delta-like 1 proteins were expressed in all tissues examined. Present findings show divergent expression of Notch 1 in skin cancer, depending on anatomical site and tumor histotype. Thus, whereas in UV-related squamous cell photocarcinogenesis Notch 1 downregulation could mirror a tumor suppressor function of the receptor, in sun-protected squamous cell carcinomas Notch 1 was upregulated. Furthermore, Notch 1 expression was minimal in basal cell carcinoma subtypes correlated with risk of recurrence (sclerodermiform/infiltrating and baso-squamous) in comparison with nodular and superficial types.

Secretion without Golgi.

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1alpha. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1alpha presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders.

A ribonuclease protection assay-based approach for analysis of angiogenic gene expression in archival tissues.

Archival, formalin-fixed and paraffin-embedded tissues routinely stored in pathology departments represent an invaluable resource for retrospective molecular biology studies for diagnostic and prognostic purposes. In such specimens extraction of transcriptionally competent RNA to be analyzed by conventional techniques, such as reverse transcription-polymerase chain reaction, is a challenging task. Therefore, we developed a novel methodological approach that allows successful detection and semiquantitative analysis of specific mRNAs obtained from archival formalin-fixed, paraffin-embedded specimens by ribonuclease protection assay. Specifically, we measured a panel of 7 angiogenic markers in selected archival tissues stored at room temperature and retrieved over a wide time span (10 y). The study series consisted in samples of benign and malignant melanocytic lesions. In our model, expression of FLT-1, the vascular-endothelial growth factor receptor-1, correlated with the expression of mRNAs encoding other tyrosine kinase receptors, such as TIE-1 and TIE-2, as well as with angiopoietin and with the protease-activated receptor-1 and vascular-endothelial growth factor itself. Relative to control (normal skin), in melanoma the expression of the selected angiogenic markers was significantly higher. In conclusion, our study provides evidence that ribonuclease protection assay on archival specimens would be highly valuable for retrospective studies, for diagnosis or prognosis.

Alpha,beta-unsaturated aldehydes in cigarette smoke release inflammatory mediators from human macrophages.

Smoking cigarettes is the major risk factor for chronic obstructive pulmonary disease (COPD). COPD is a condition associated with chronic pulmonary inflammation, characterized by macrophage activation, neutrophil recruitment, and cell injury. Many substances contained in cigarette smoke, including reactive oxygen species (ROS), have been proposed to be responsible for the inflammatory process of COPD. However, this issue remains unsettled. By gas chromatography/mass spectrometry (GC/MS) we show that acrolein and crotonaldehyde, two alpha,beta-unsaturated aldehydes, are contained in aqueous cigarette smoke extract (CSE) at micromolar concentrations and mimic CSE in evoking the release of the neutrophil chemoattractant IL-8 and of the pleiotropic inflammatory cytokine TNF-alpha from the human macrophagic cell line U937. In addition, acrolein (10-30 microM) released IL-8 also from cultured human alveolar macrophages and THP-1 macrophagic cells. 4-hydroxy-2-nonenal (30-100 microM), an endogenous alpha,beta-unsaturated aldehyde that is abundant in lungs of patients with COPD, stimulated the release of IL-8 from U937 cells, whereas the saturated aldehyde, acetaldehyde, was ineffective. CSE-evoked IL-8 release was remarkably (> 80%) inhibited by N-acetyl-cysteine (0.1-3 mM) or glutathione monoethyl ester (1-3 mM). Both compounds, by forming covalent adducts (Michael adducts), completely removed unsaturated aldehydes from CSE. Our data demonstrate that alpha,beta-unsaturated aldehydes are major mediators of cigarette smoke-induced macrophage activation, and suggest that they might contribute to pulmonary inflammation associated with cigarette smoke.