HMGB1-Induced Cross Talk between PTEN and miRs 221/222 in Thyroid Cancer

High mobility group box 1 (HMGB1) is an ubiquitous protein that plays different roles in the nucleus, cytoplasm and extra-cellular space. It is an important DAMP molecule that allows communication between damaged or tumor cells and the immune system. Tumor cells exploit HMGB1’s ability to activate intracellular pathways that lead to cell growth and migration. Papillary thyroid cancer is a well differentiated tumor and is often used to study relationships between cells and the inflammatory microenvironment as the latter is characterized by high levels of inflammatory cells and cytokines. Anaplastic thyroid cancer is one of the most lethal human cancers in which many microRNAs and tumor suppressor genes are de-regulated. Up-regulation of microRNAs 221 and 222 has been shown to induce the malignant phenotype in many human cancers via inhibition of PTEN expression. In this study we suggest that extracellular HMGB1 interaction with RAGE enhances expression of oncogenic cluster miR221/222 that in turn inhibits tumor suppressor gene PTEN in two cell lines derived from human thyroid anaplastic and papillary cancers. The newly identified pathway HMGB1/RAGE/miR 221/222 may represent an effective way of tumor escape from immune surveillance that could be used to develop new therapeutic strategies against anaplastic tumors

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

BACKGROUND: Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. METHODS: The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®). RESULTS: Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively. CONCLUSIONS: We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms

Collagen-induced Platelet Shape Change Is Not Affected by Positive Feedback Pathway Inhibitors and cAMP-elevating Agents

Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK

Aspirin-dependent effects on purinergic P2Y1 receptor express

Chronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y1 receptor exerts its effects via a Gq-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y1 expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y1 mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y1 mRNA expression was higher than in mock culture. P2Y1 expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y1 expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y1 up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y1 is coupled to the same Gq-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway

From human Megakaryocytes to platelets: Effects of aspirin on high-mobility group Box 1/receptor for advanced glycation end products axis

Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of in vitro differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin “in vivo” and “in vitro” not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1

Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation

AbstractIt has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (Gi and Gq) is essential for platelet aggregation: in fact, the thromboxane A2 synthetic agonist, U46619, which causes the selective activation of Gq, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct Gq activation is not required for platelet aggregation and that the activation of an enzyme downstream of Gq, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCγ2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a Gi protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a Gi protein-coupled receptor is activated

Distinct platelet crosstalk with adaptive and innate immune cells after adenoviral and mRNA vaccination against SARS-CoV-2

Background: Genetic-based COVID-19 vaccines have proved highly effective in reducing the risk of hospitalization and death. As they were first distributed on a large-scale population, adenoviral-based vaccines were linked to a very rare thrombosis with thrombocytopenia syndrome and the interplay between platelets and vaccinations increasingly gained attention. Objective: To study the crosstalk between platelets and the vaccine-induced immune response. Methods: We prospectively enrolled young healthy volunteers who received the mRNA-based vaccine, BNT162b2 (n=15), or the adenovirus-based vaccine, AZD1222 (n=25) and studied their short-term platelet and immune response before and after vaccine injections. In a separate cohort, we retrospectively analysed the effect of aspirin on the antibody response 1 and 5 months after BNT162b2 vaccination. Results: Here we show that a faster antibody response to either vaccine is associated to the formation of platelet aggregates with marginal zone-like B-cells, a subset geared to bridge the temporal gap between innate and adaptive immunity. However, while the mRNA-based vaccine is associated with a more gradual and tolerogenic response that fosters the crosstalk between platelets and adaptive immunity, the adenovirus-based vaccine, the less immunogenic of the two, evokes an antiviral-like response during which platelets are cleared and less likely to cooperate with B-cells. Moreover, subjects taking aspirin (n=56) display lower antibody levels after BNT162b2 vaccination compared to matched individuals. Conclusions: Platelets are a component of the innate immune pathways that promote the B-cell response after vaccination. Future studies on the platelet-immune crosstalk post-immunization will improve safety, efficacy, and strategic administration of next-generation vaccines

Failure of apoptosis and activation on NFκB by celecoxib and aspirin in lung cancer cell lines

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SARS-CoV-2 infection predicts larger infarct volume in patients with acute ischemic stroke

Background and purpose: Acute ischemic stroke (AIS) is a fearful complication of Coronavirus Disease-2019 (COVID-19). Aims of this study were to compare clinical/radiological characteristics, endothelial and coagulation dysfunction between acute ischemic stroke (AIS) patients with and without COVID-19 and to investigate if and how the SARS-CoV-2 spike protein (SP) was implicated in triggering platelet activation. Methods: We enrolled AIS patients with COVID-19 within 12 h from onset and compared them with an age- and sex-matched cohort of AIS controls without COVID-19. Neuroimaging studies were performed within 24 h. Blood samples were collected in a subset of 10 patients. Results: Of 39 AIS patients, 22 had COVID-19 and 17 did not. Admission levels of Factor VIII and von Willebrand factor antigen were significantly higher in COVID-19 patients and positively correlated with the infarct volume. In multivariate linear regression analyses, COVID-19 was an independent predictor of infarct volume (B 20.318, Beta 0.576, 95%CI 6.077-34.559; p = 0.011). SP was found in serum of 2 of the 10 examined COVID-19 patients. Platelets from healthy donors showed a similar degree of procoagulant activation induced by COVID-19 and non-COVID-19 patients' sera. The anti-SP and anti-FcγRIIA blocking antibodies had no effect in modulating platelet activity in both groups. Conclusions: SARS-CoV-2 infection seems to play a major role in endothelium activation and infarct volume extension during AIS