Isolation and assessment of the in vitro anti-tumor activity of smenothiazole A and B, chlorinated thiazole-containing peptide/polyketides from the Caribbean sponge, Smenospongia aurea.

The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.

AurkA inhibitors enhance the effects of B-RAF and MEK inhibitors in melanoma treatment.

BACKGROUND: Aurora kinase A (AurkA) is over-expressed in melanoma and its inhibition has been observed to limit tumor growth, suggesting a potential role in melanoma treatment. METHODS: A human melanoma cell line with the B-RAF (V600E) mutation (A375mel) was exposed to B-RAF inhibitor (GSK2118436), MEK inhibitor (GSK1120212) and AurkA inhibitor (MLN8054) as single agents or in various combinations (BRAF plus AurkA inhibitor, MEK plus AurkA inhibitor or triple combination BRAF plus MEK plus AurkA inhibitor). Cell proliferation was assessed using xCELLigence technology. Total protein extracts were examined for p53 and c-Myc protein expression by Western blot analysis. Drug anti-tumor effects were further assessed using a 3D-human melanoma skin reconstruction model, in which tissues were incubated with serum-free medium containing control, B-RAF plus MEK inhibitor, MEK plus AurkA inhibitor or the triple combination. RESULTS: AurkA inhibitor plus B-RAF inhibitor, AurkA inhibitor plus MEK inhibitor or triple combination had a markedly greater anti-proliferative effect on A375 (BRAFV600E) melanoma cells than single agents. In the 3D human skin model, the triple combination had a greater anti-tumor effect at the epidermal/dermal junction than control or either double combination. However, S-100 and Ki-67 positively stained spindle-shaped cells were detected in the dermal stratum, suggesting the presence of alive and proliferating melanoma cells. CONCLUSIONS: These findings provide new prospects for melanoma research, including combined B-RAF/AurkA inhibition for B-RAF mutated melanomas and MEK/AurkA inhibitor combination for patients without B-RAF mutations. Moreover, for the first time, we have shown that a B-RAF, MEK and AurkA inhibitor triple drug combination offers increased efficacy against melanoma cell growth and might be considered as a potential treatment strategy for enhancing clinical response in melanoma. However, although this triple drug combination was more effective at the epidermal/dermal junction, the suggested presence of alive and proliferating melanoma cells in the dermal stratum could result in drug resistance and disease recurrence. Molecular characterization of these dermal cells may be critical for the development of novel therapeutic strategies.

Left Heart Disease Phenotype in Elderly Patients with Pulmonary Arterial Hypertension: Insights from the Italian PATRIARCA Registry.

Pulmonary arterial hypertension (PAH) in the elderly is often associated with left heart disease (LHD), prompting concerns about the use of pulmonary vasodilators. The PATRIARCA registry enrolled ≥70 year-old PAH or chronic thromboembolic pulmonary hypertension (CTEPH) patients at 11 Italian centers from 1 December 2019 through 15 September 2022. After excluding those with CTEPH, post-capillary PH at the diagnostic right heart catheterization (RHC), and/or incomplete data, 23 (33%) of a total of 69 subjects met the criteria proposed in the AMBITION trial to suspect LHD. Diabetes [9 (39%) vs. 6 (13%), p = 0.01] and chronic kidney disease [14 (61%) vs. 12 (26%), p = 0.003] were more common, and the last RHC pulmonary artery wedge pressure [14 ± 5 vs. 10 ± 3 mmHg, p < 0.001] was higher and pulmonary vascular resistance [5.56 ± 3.31 vs. 8.30 ± 4.80, p = 0.02] was lower in LHD than non-LHD patients. However, PAH therapy was similar, with 13 (57%) and 23 (50%) subjects, respectively, taking two oral drugs. PAH medication patterns remained comparable between LHD and non-LHD patients also when the former [37, 54%] were identified by atrial fibrillation and echocardiographic features of LHD, in addition to the AMBITION criteria. In this real-world snapshot, elderly PAH patients were treated with pulmonary vasodilators, including combinations, despite a remarkable prevalence of a LHD phenotype.

Antioxidant and anti-proliferative properties of extracts from heterotrophic cultures of Galdieria sulphuraria.

This study explores the possibility to use the extremophilic microalga Galdieria sulphuraria (strain 064) as a source of natural biomolecules with beneficial and protective effects on human health. Galdieria was cultivated in heterotrophy conditions and cells extracts for their antioxidant and anti-proliferative properties were tested. Galdieria extracts showed high antioxidant power tested through ABTS assay and revealed high glutathione and phycocyanin contents. Based on Annexin-V FITC/propidium iodide and MTT analysis, algae extracts inhibited the proliferation of human adenocarcinoma A549 cells (51.2% inhibition) through the induction of apoptosis without cell cycle arrest. Besides, cytotoxicity and cytometry assays showed a positive pro-apoptotic mechanism. On these bases, we suggest that G. sulphuraria from heterotrophic culture, for its therapeutic potential, could be considered a good candidate for further studies with the aim to isolate bioactive anti-cancer molecules.

Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-ß1 quantification. We found that LC212-CM contains high levels of TGF-ß1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-ß1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-ß1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-ß1. In conclusion, hsa-miR21 and TGF-ß1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells.

Causes and impact on survival of underuse of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in heart failure.

Guidelines recommend angiotensin-converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) for treatment of heart failure with reduced ejection fraction (HFrEF), but these medications are underprescribed in clinical practice. We reviewed the records of HF patients receiving a first visit in a tertiary outpatient clinic from January 1st 2004 to May 31st 2015, and selected those with a serum creatinine concentration (sCr) available at both the first and last visit and < 3.5 mg/dL at baseline, and a left ventricular ejection fraction (LVEF) < 50% at the first visit. Of 570 eligible patients, 92 (16.1%) never received ACEi/ARB. Compared to ACEi/ARB users, never-users were older, more often women, had higher sCr and lower systolic blood pressure, were less commonly on beta-blocker, and had more frequently anemia. Current or prior cancer also tended to be more common in ACEi/ARB never-users. ACEi/ARB users displayed an improvement in LVEF by ≥ 10% of the baseline value more often than ACEi/ARB never-users (33.7% vs. 20.7%, respectively, P = 0.01), whereas no difference in percent variation of sCr levels was found between the two groups (8.2% vs. 3.1%, respectively; P = 0.13). Over a median follow-up of 56 months (range 1-137 months), 215 (37.7%) patients died. After multiple adjustments, ACEi/ARB never-use was associated with an almost twofold increased risk of all-cause mortality (HR 1.97, 95%CI 1.39-2.80). ACEi/ARB underuse in HFrEF is a standing issue with dramatic prognostic consequences. Efforts are needed to eliminate perceived contraindications to these drugs and ensure their implementation in real-life cardiology.

Clinical Characteristics and Long-Term Mortality Rate in Female Patients with Takotsubo Syndrome Compared with Female Patients with ST-Elevation Acute Myocardial Infarction: A Retrospective Study from a Single Center.

BACKGROUND: Takotsubo syndrome (TTS) is characterized by acute transient, stress-induced, left ventricular systolic dysfunction, generally presenting with apical ballooning. It can mimic an acute coronary syndrome, but with a milder increase in cardiac enzymes and without culprit coronary artery disease on angiography. Data on long-term follow-up and survival in patients with TTS, compared with patients with ST-elevation myocardial infarction (STEMI), are scarce. PURPOSE: To assess all-cause mortality rate and survival in a consecutive series of female patients with TTS compared with age- and sex-matched STEMI patients on long-term follow-up. METHODS AND RESULTS: We collected data of 65 TTS female patients (TTS group) with a mean age of 73.42 ± 11.35 years from 2001 to 2013. Collection of follow-up information was concluded for all patients in 2016. To compare the mortality and survival of TTS patients with those of the STEMI population, we used data from our STEMI Registry, a prospective registry of 7446 STEMI patients admitted from 2001 to 2013 to our cath-lab for primary percutaneous coronary intervention (p-PCI). From the registry, we selected 104 STEMI patients (STEMI group) comparable to our TTS group in terms of age (mean age of 72.33 ± 11.92 years) and sex. On follow-up examination after a median of 1000 days, the TTS group had a lower all-cause mortality rate than the STEMI group (7.69% versus 23.08%). This difference was statistically different between the two groups (log-rank test, p value = 0.03). CONCLUSIONS: In our study, TTS and STEMI patients displayed a statistically significant difference in long-term survival. Specifically, the TTS group had a lower mortality rate than the STEMI group. This seems to suggest that TTS and STEMI are two different clinical entities with two different clinical outcomes.

Methods, accuracy and clinical implications of atrial fibrillation detection by cardiac implantable electronic devices.

Atrial fibrillation (AF) is the most common arrhythmia in the developed countries and is associated with an increased risk of thromboembolic events and heart failure. Episodes of AF are often asymptomatic and intermittent, eluding diagnosis with non-continuous monitoring techniques. Cardiac implantable electronic devices (CIEDs) represent the gold standard for detecting asymptomatic AF. Improper CIED programming may however increase the risk of false-positive detection of atrial tachyarrhythmias, leading to inappropriate clinical management of patients. A faster rate and a longer duration of the tachyarrhythmic episodes, in addition to a greater AT/AF burden, have been proposed as potential criteria for differentiating between CIED-detected atrial tachyarrhythmias and true AF. Nonetheless, manual overreading of intracardiac electrograms recorded by the CIED remains crucial for a correct diagnosis. Asymptomatic atrial tachyarrhythmias may carry a higher risk of systemic thromboembolism, though clinical thromboembolic risk factors seem to play a greater if not absolute role in prognostication. In addition, there is no clear temporal relationship between CIED-detected atrial tachyarrhythmias and stroke, and the anticoagulation strategy to be pursued in these patients is still a matter of debate and the focus of current prospective randomized studies.

Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.

Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5ß1 and ανß3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5ß1 and ανß3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5ß1 or ανß3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5ß1 or ανß3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5ß1 and ανß3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.

Yin Yang I as an Epimodulator of miRNAs in the Metastatic Cascade.

Yin Yang 1 (YY1) belongs to the polycomb group (PcG) of proteins that modify chromatin epigenetically during dynamic regulation of their target genes. The predominant feature of YY1 is the zinc finger, an ancient structural motif that mediates protein-protein interactions and is capable of interacting with both DNA and RNA. Evidence reveals that YY1 acts predominantly as an epigenetic modulator, influencing the activity and/or localization of epigenetic modifiers molecules such as DNA methylation transferases, histone deacetylases, or non-coding RNAs. Deregulation of the epigenome is observed frequently in a variety of cancer types and is often correlated directly with advanced metastatic stages and poor prognosis. In this review, we address the current understanding of YY1 as a recruiter of epi-modifier molecules in the mechanism of aberrant regulation of target genes as a part of the metastatic cascade.

Post-surgery fluids promote transition of cancer stem cell-to-endothelial and AKT/mTOR activity, contributing to relapse of giant cell tumors of bone.

Giant cell tumors of bone (GCTB) are rare sarcomas with a high rate of unpredictable local relapse. Studies suggest that surgical methods affect recurrence, supporting the idea that local disease develops from re-growth of residual cancer cells. To identify early prognostic markers of individual risk of recurrence, we evaluated the effect of post-surgery fluids from a cohort of GCTB patients on growth of primary and established sarcoma cell lines, and mice xenograph. Post-surgery fluids increased cell growth and enhanced expression of CD44++, the principal receptor for the extracellular matrix component hyaluronan and the mesenchymal stem marker CD117+. Cancer cells became highly invasive and tumorigenic, acquiring stemness properties, and activated AKT/mTOR pathway. Prolonged stimulation with post-surgery fluids down-regulated the mesenchymal gene TWIST1 and Vimentin protein, and transdifferentiated cells into tubule-like structures positive to the endothelial markers VE-Cadherin and CD31+. In mice, post-surgery fluids gave rise to larger and more vascularized tumors than control, while in patients AKT/mTOR pathway activation was associated with recurrence by logistic regression (Kaplan-Meier; P<0.001). These findings indicate that post-surgery fluids are an adjuvant in mechanisms of tumor regrowth, increasing stem cell growth and AKT/mTOR activity.

Clinical characteristics and prognostic impact of atrial fibrillation in patients with chronic heart failure.

AIM: To assess the prevalence, clinical characteristics and independent prognostic impact of atrial fibrillation (AF) in chronic heart failure (CHF) patients, and the potential protective effect of disease-modifying medications, particularly beta-blockers (BB). METHODS: We retrospectively reviewed the charts of patients referred to our center since January 2004, and collected all clinical information available at their first visit. We assessed mortality to the end of June 2015. We compared patients with and without AF, and assessed the association between AF and all-cause mortality by multivariate Cox regression and Kaplan-Meyer analysis, particularly accounting for ongoing treatment with BB. RESULTS: A total of 903 patients were evaluated (mean age 68 ± 12 years, 73% male). Prevalence of AF was 19%, ranging from 10% to 28% in patients ≤ 60 and ≥ 77 years, respectively. Besides the older age, patients with AF had more symptoms (New York Heart Association II-III 60% vs 44%), lower prevalence of dyslipidemia (23% vs 37%), coronary artery disease (28% vs 52%) and left bundle branch block (9% vs 16%). On the contrary, they more frequently presented with an idiopathic etiology (50% vs 24%), a history of valve surgery (13% vs 4%) and received overall more devices implantation (31% vs 21%). The use of disease-modifying medications (i.e., BB and ACE inhibitors/angiotensin receptor blockers) was lower in patients with AF (72% vs 80% and 71% vs 79%, respectively), who on the contrary were more frequently treated with symptomatic and antiarrhythmic drugs including diuretics (87% vs 69%) and digoxin (51% vs 11%). At a mean follow-up of about 5 years, all-cause mortality was significantly higher in patients with AF as compared to those in sinus rhythm (SR) (45% vs 34%, P value < 0.05 for all previous comparisons). However, in a multivariate analysis including the main significant predictors of all-cause mortality, the univariate relationship between AF and death (HR = 1.49, 95%CI: 1.15-1.92) became not statistically significant (HR = 0.98, 95%CI: 0.73-1.32). Nonetheless, patients with AF not receiving BB treatment were found to have the worst prognosis, followed by patients with SR not receiving BB therapy and patients with AF receiving BB therapy, who both had similarly worse survival when compared to patients with SR receiving BB therapy. CONCLUSION: AF was highly prevalent and associated with older age, worse clinical presentation and underutilization of disease-modifying medications such as BB in a population of elderly patients with CHF. AF had no independent impact on mortality, but the underutilization of BB in this group of patients was associated to a worse long-term prognosis.