In Hcmv-Exposed Huvec, P52/Relb Regulatory Factors Mediate Activation of the Human Par1 Gene Promoter

We studied the effect of HCMV (AD-169) on the regulation of PAR1 reporter activity in HUVEC. In HUVEC, HCMV significantly induced activation of PAR1 reporter constructs carrying AP-2-like and two complexes of AP-2/Sp1-binding elements. Shorter constructs with no AP-2-like binding elements had lower activity upon virus inoculation. Moreover, activity of the shortest construct without binding elements was not significantly increased in virus-exposed cells. Furthermore, in HCMV-exposed cells, activity of the PARI reporter was significantly augmented upon p52/RelB transfection. Taken together, the data indicate that activity of the PAR1 promoter is highly dependent on AP-2/Sp1 binding elements and mediated by p52/RelB regulatory factors

Expression and function of protease-activated receptors in human monocyte-derived dendritic cells

Protease-activated receptors (PARs) are activated by proteolytic cleavage of their extracellular domain, unmasking a new N-terminus acting as a tethered ligand. PAR1 and PAR3 were investigated at the promoter level. Using DNase I footprinting, binding of the AP-1 transcription factor to the PAR1 promoter was localized. This indicates that NF-kB does not activate the promoter directly, but interacts with AP-1, which then occupies its binding site on the promoter. The role of PARs in human monocyte-derived dendritic cells was further investigated. Three different inducers of dendritic cell maturation were used, LPS, TNF-a, and CD40L. LPS maturation elicited enhanced expression of PAR1 and PAR3, while the two other stimuli slightly up-regulated PAR1 and PAR3 expression. PAR4 expression remained undetectable in all three maturation types. Thrombin stimulation of LPS-matured dendritic cells resulted in a chemotactic response. The peptide agonists, PAR1-AP and PAR3-AP, produced thrombin-like effects on chemotaxis. PAR1 and PAR3 antibodies were effective in blocking the thrombin responses. Subsequently we investigated the thrombin signalling pathway in dendritic cells, and detected that thrombin activates the Rho-kinase (ROCK), which phosphorylates the myosin light chain (MLC) 2. Thrombin stimulation also induced an increase in actin polymerisation. Both of these events are important for the cell migration. Finally, the treatment of dendritic cells with the specific pharmacological inhibitor of ROCK (Y-27632), completely abolished the MLC phosphorylation, suggesting that thrombin stimulates the dendritic cell migration through Rho/ROCK pathway. The major finding of this work is that dendritic cells are activated by thrombin. Since both thrombin and dendritic cells are present in atherosclerotic plaques, targeting this mechanism in the endothelium could prevent plaque destabilisation and become a novel therapeutic strategy for the treatment of atherosclerosis

Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1 alpha and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation

Malignant Risk Stratification of Thyroid FNA Specimens With Indeterminate Cytology Based on Molecular Testing

BACKGROUND: Fine-needle aspiration (FNA) has been employed for many years for examining thyroid nodules, and the cytology of aspirates is the primary determinant for whether thyroidectomy is indicated. Fifteen to thirty percent of thyroid nodules, not being clearly benign or malignant, fall into an indeterminate category. The main goals of molecular diagnostics for thyroid nodules are to prevent unnecessary surgery in patients with benign nodules and to stop patients with malignant nodules from being subjected to repeated operations. METHODS: This study was designed to evaluate the diagnostic utility of 4 markers in thyroid FNA cytology via testing for the BRAF V600E mutation and the expression of microRNA-221, microRNA-222, and galectin-3 protein in FNA samples with indeterminate cytology. RESULTS: A predictor model distinguishing benign samples from malignant samples on the basis of the 4 aforementioned markers was formulated. This decision model provided a sensitivity of 73.5%, a specificity of 89.8%, and a diagnostic accuracy of 75.7%. The positive predictive value was 80.6%, and the negative predictive value was 85.5%; this suggested that the prediction had good reliability. CONCLUSIONS: One hundred twenty FNA samples were examined, and 62 nodules were classified as benign with the proposed diagnostic algorithm. This resulted in a reduction of the initial 120 patients to 58 and thus decreased by half the number of persons undergoing surgery

Carboranyl Analogues of Ketoprofen with Cytostatic Activity against Human Melanoma and Colon Cancer Cell Lines

Ketoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) that also exhibits cytotoxic activity against various cancers. This makes ketoprofen an attractive structural lead for the development of new NSAIDs and cytotoxic agents. Recently, the incorporation of carboranes as phenyl mimetics in structures of established drugs has emerged as an attractive strategy in drug design. Herein, we report the synthesis and evaluation of four novel carborane-containing derivatives of ketoprofen, two of which are prodrug esters with an nitric oxide-releasing moiety. One of these prodrug esters exhibited high cytostatic activity against melanoma and colon cancer cell lines. The most pronounced activity was found in cell lines that are sensitive to oxidative stress, which was apparently induced by the ketoprofen analogue

The More the Better—Investigation of Polymethoxylated <i>N</i>-Carboranyl Quinazolines as Novel Hybrid Breast Cancer Resistance Protein Inhibitors

The ineffectiveness and failing of chemotherapeutic treatments are often associated with multidrug resistance (MDR). MDR is primarily linked to the overexpression of ATP-binding cassette (ABC) transporter proteins in cancer cells. ABCG2 (ATP-binding cassette subfamily G member 2, also known as the breast cancer resistance protein (BCRP)) mediates MDR by an increased drug efflux from the cancer cells. Therefore, the inhibition of ABCG2 activity during chemotherapy ought to improve the efficacy of the administered anti-cancer agents by reversing MDR or by enhancing the agents’ pharmacokinetic properties. Significant efforts have been made to develop novel, powerful, selective, and non-toxic inhibitors of BCRP. However, thus far the clinical relevance of BCRP-selective MDR-reversal has been unsuccessful, due to either adverse drug reactions or significant toxicities in vivo. We here report a facile access towards carboranyl quinazoline-based inhibitors of ABCG2. We determined the influence of different methoxy-substitution patterns on the 2-phenylquinazoline scaffold in combination with the beneficial properties of an incorporated inorganic carborane moiety. A series of eight compounds was synthesized and their inhibitory effect on the ABCG2-mediated Hoechst transport was evaluated. Molecular docking studies were performed to better understand the structure-protein interactions of the novel inhibitors, exhibiting putative binding modes within the inner binding site. Further, the most potent, non-toxic compounds were investigated for their potential to reverse ABCG2-mediated mitoxantrone (MXN) resistance. Of these five evaluated compounds, N-(closo-1,7-dicarbadodecaboran(12)-9-yl)-6,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-quinazolin-4-amine (DMQCd) exhibited the strongest inhibitory effect towards ABCG2 in the lower nanomolar ranges. Additionally, DMQCd was able to reverse BCRP-mediated MDR, making it a promising candidate for further research on hybrid inorganic-organic compounds

Carboranyl Analogues of Celecoxib with Potent Cytostatic Activity against Human Melanoma and Colon Cancer Cell Lines.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common way of treating inflammatory disorders. Their widespread use helped reveal their other modes of action as pharmaceuticals, such as a profound effect on various cancers. Celecoxib has proven to be a very prominent member of this group with cytostatic activities. On the other hand, the highly dynamic field of drug design is constantly searching for new ways of modifying known structures to obtain more powerful and less harmful drugs. A very interesting development is the implementation of carboranes in pharmacologically active structures, mostly as phenyl mimetics. Herein we report the synthesis of three carborane-containing derivatives of the COX-2-selective NSAID celecoxib. The new compounds proved to have promising cytostatic potential against various melanoma and colorectal adenocarcinoma cell lines. Inhibited proliferation accompanied by caspase-independent apoptotic cell death was found to be the main cause of decreased cell viability upon treatment with the most efficient celecoxib analogue, 3 b (4-[5-(1,7-dicarba-closo-dodecaboranyl)-3-trifluoromethyl-1H-pyrazol-1-yl]-1-methylsulfonylbenzene)

Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease

We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F(2) crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload-induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease