Role of Hypoxia and Rac1 Inhibition in the Metastatic Cascade.

The hypoxic condition has a pivotal role in solid tumors and was shown to correlate with the poor outcome of anticancer treatments. Hypoxia contributes to tumor progression and leads to therapy resistance. Two forms of a hypoxic environment might have relevance in tumor mass formation: chronic and cyclic hypoxia. The main regulators of hypoxia are hypoxia-inducible factors, which regulate the cell survival, proliferation, motility, metabolism, pH, extracellular matrix function, inflammatory cells recruitment and angiogenesis. The metastatic process consists of different steps in which hypoxia-inducible factors can play an important role. Rac1, belonging to small G-proteins, is involved in the metastasis process as one of the key molecules of migration, especially in a hypoxic environment. The effect of hypoxia on the tumor phenotype and the signaling pathways which may interfere with tumor progression are already quite well known. Although the role of Rac1, one of the small G-proteins, in hypoxia remains unclear, predominantly, in vitro studies performed so far confirm that Rac1 inhibition may represent a viable direction for tumor therapy.

Dual Inhibitors of AChE and BACE-1 for Reducing Aß in Alzheimer's Disease: From In Silico to In Vivo.

Alzheimer's disease (AD) is a complex and widespread condition, still not fully understood and with no cure yet. Amyloid beta (Aß) peptide is suspected to be a major cause of AD, and therefore, simultaneously blocking its formation and aggregation by inhibition of the enzymes BACE-1 (ß-secretase) and AChE (acetylcholinesterase) by a single inhibitor may be an effective therapeutic approach, as compared to blocking one of these targets or by combining two drugs, one for each of these targets. We used our ISE algorithm to model each of the AChE peripheral site inhibitors and BACE-1 inhibitors, on the basis of published data, and constructed classification models for each. Subsequently, we screened large molecular databases with both models. Top scored molecules were docked into AChE and BACE-1 crystal structures, and 36 Molecules with the best weighted scores (based on ISE indexes and docking results) were sent for inhibition studies on the two enzymes. Two of them inhibited both AChE (IC50 between 4-7 µM) and BACE-1 (IC50 between 50-65 µM). Two additional molecules inhibited only AChE, and another two molecules inhibited only BACE-1. Preliminary testing of inhibition by F681-0222 (molecule 2) on APPswe/PS1dE9 transgenic mice shows a reduction in brain tissue of soluble Aß42.

Syndecan-1 Promotes Hepatocyte-Like Differentiation of Hepatoma Cells Targeting Ets-1 and AP-1.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which is indispensable in the structural and functional integrity of epithelia. Normal hepatocytes display strong cell surface expression of syndecan-1; however, upon malignant transformation, they may lose it from their cell surfaces. In this study, we demonstrate that re-expression of full-length or ectodomain-deleted syndecan-1 in hepatocellular carcinoma cells downregulates phosphorylation of ERK1/2 and p38, with the truncated form exerting an even stronger effect than the full-length protein. Furthermore, overexpression of syndecan-1 in hepatoma cells is associated with a shift of heparan sulfate structure toward a highly sulfated type specific for normal liver. As a result, cell proliferation and proteolytic shedding of syndecan-1 from the cell surface are restrained, which facilitates redifferentiation of hepatoma cells to a more hepatocyte-like phenotype. Our results highlight the importance of syndecan-1 in the formation and maintenance of differentiated epithelial characteristics in hepatocytes partly via the HGF/ERK/Ets-1 signal transduction pathway. Downregulation of Ets-1 expression alone, however, was not sufficient to replicate the phenotype of syndecan-1 overexpressing cells, indicating the need for additional molecular mechanisms. Accordingly, a reporter gene assay revealed the inhibition of Ets-1 as well as AP-1 transcription factor-induced promoter activation, presumably an effect of the heparan sulfate switch.

Chemotherapy induced PRL3 expression promotes cancer growth via plasma membrane remodeling and specific alterations of caveolae-associated signaling.

BACKGROUND: The outcome of cancer therapy is greatly defined by the ability of a tumor cell to evade treatment and re-establish its bulk mass after medical interventions. Consequently, there is an urgent need for the characterization of molecules affecting tumor reoccurrence. The phosphatase of regenerating liver 3 (PRL3) protein was recently emerged among the targets that could affect such a phenomenon. METHODS: The expression induction of PRL3 in melanoma cells treated with chemotherapeutic agents was assessed by western blotting. The effect of PRL3 expression on cancer growth was investigated both in vitro and in vivo. The association of PRL3 with the caveolae structures of the plasma membrane was analyzed by detergent free raft purification. The effect of PRL3 expression on the membrane organization was assayed by electron microscopy and by membrane biophysical measurements. Purification of the plasma membrane fraction and co-immunoprecipitation were used to evaluate the altered protein composition of the plasma membrane upon PRL3 expression. RESULTS: Here, we identified PRL3 as a genotoxic stress-induced oncogene whose expression is significantly increased by the presence of classical antitumor therapeutics. Furthermore, we successfully connected the presence of this oncogene with increased tumor growth, which implies that tumor cells can utilize PRL3 effects as a survival strategy. We further demonstrated the molecular mechanism that is connected with the pro-growth action of PRL3, which is closely associated with its localization to the caveolae-type lipid raft compartment of the plasma membrane. In our study, PRL3 was associated with distinct changes in the plasma membrane structure and in the caveolar proteome, such as the dephosphorylation of integrin ß1 at Thr788/Thr789 and the increased partitioning of Rac1 to the plasma membrane. These alterations at the plasma membrane were further associated with the elevation of cyclin D1 in the nucleus. CONCLUSIONS: This study identifies PRL3 as an oncogene upregulated in cancer cells upon exposure to anticancer therapeutics. Furthermore, this work contributes to the existing knowledge on PRL3 function by characterizing its association with the caveolae-like domains of the plasma membrane and their resident proteins.

Cell type-dependent HIF1 α-mediated effects of hypoxia on proliferation, migration and metastatic potential of human tumor cells.

Tumor hypoxia promotes neoangiogenesis and contributes to the radio- and chemotherapy resistant and aggressive phenotype of cancer cells. However, the migratory response of tumor cells and the role of small GTPases regulating the organization of cytoskeleton under hypoxic conditions have yet to be established. Accordingly, we measured the proliferation, migration, RhoA activation, the mRNA and protein levels of hypoxia inducible factor-1alpha (HIF-1α) and three small G-proteins, Rac1, cdc42 and RhoA in a panel of five human tumor cell lines under normoxic and hypoxic conditions. Importantly, HT168-M1 human melanoma cells with high baseline migration capacity showed increased HIF-1α and small GTPases expression, RhoA activation and migration under hypoxia. These activities were blocked by anti- HIF-1α shRNA. Moreover, the in vivo metastatic potential was promoted by hypoxia mimicking CoCl2 treatment and reduced upon inhibition of HIF-1α in a spleen to liver colonization experiment. In contrast, HT29 human colon cancer cells with low migration capacity showed limited response to in vitro hypoxia. The expression of the small G-proteins decreased both at mRNA and protein levels and the RhoA activation was reduced. Nevertheless, the number of lung or liver metastatic colonies disseminating from orthotopic HT29 grafts did not change upon CoCl2 or chetomin treatment. Our data demonstrates that the hypoxic environment induces cell-type dependent changes in the levels and activation of small GTPases and results in varying migratory and metastasis promoting responses in different human tumor cell lines.

Boyden chamber-based method for characterizing the distribution of adhesions and cytoskeletal structure in HT1080 fibrosarcoma cells.

A 2D model was previously presented that describes the gliding motility of human fibrosarcoma cells. The model was based on the observation that adhesions are present only on the outer rim of the leading lamella of the semicircular cell. The present model describes the organization of adhesions and the cytoskeleton of migrating HT1080 fibrosarcoma and LX2 hepatic stellate cells in three dimensions. The migration assays were performed in a modified Boyden chamber using fibronectin, Matrigel, or collagen I as chemoattractants. The distribution of the adhesions was analyzed by confocal laser scanning microscope, and following decoration with heavy meromyosin, the organization of actin filaments was analyzed by electron microscopy. Double labeling was performed to study the relationship of the actin and vimentin filament network in the moving cells. Vinculin containing adhesions were observed only at the front of the cell in the form of a ring while passing through a filter pore of the Boyden chamber. Actin filaments were present only below the plasma membrane, except the very tip of the leading lamella. Vimentin intermediate filaments were localized around the cell nucleus behind the actin filament-rich lamella. This paper describes a model of the organization of adhesions and the cytoskeleton of migrating cells in the Boyden chamber. The model is based on the observation that adhesions are present only at the leading edge of the cell. The results extend the earlier 2D model of cell locomotion into 3D.

No mutation but high mRNA expression of Coxsackie-Adenovirus Receptor was observed in both dilated and ischemic cardiomyopathy.

The most common causes of acute myocarditis and the possible consequence of dilated cardiomyopathy are virus infections. The receptor of the two most common viruses connected to these myocardial diseases was identified as Coxsackie-Adenovirus Receptor. The purpose of this study was to assess Coxsackie-Adenovirus Receptor mRNA expression in the myocardium and search for mutations in the Coxsackie-Adenovirus Receptor gene to compare dilated, inflammatory and ischemic cardiomyopathy with control group. All the myocardial samples were obtained from 35 explanted hearts during heart transplantation, than DNA and RNA were isolated from the muscle samples. cDNA was generated from RNA using reverse transcription, and real-time PCR was performed with relative quantification by ß-actin gene as endogenous control. Using DNA extracted from the myocardial samples, we sequenced all the seven exons of the Coxsackie-Adenovirus Receptor gene. Coxsackie-Adenovirus Receptor mRNA expression was higher in both ischemic and dilated cardiomyopathy groups than in inflammatory cardiomyopathy and healthy control groups. Sequencing of CAR gene showed no sign of mutation. Therefore, the sequences result of CAR exons did not show any mutation or polymorphism, that explains a determinant role of CAR in dilated or ischemic CM. Our results suggest that high mRNA expression of Coxsackie-Adenovirus Receptor may support its role in regeneration of the damaged myocardium rather than having any role in viral mediated heart disease.

The role of viral infections in the development of dilated cardiomyopathy.

Enteroviruses (EVs) are the most frequent pathogens in myocarditis and in the subsequently developing dilated cardiomyopathy as well. Furthermore, persistence of other viruses might play a pathogenic role in the evolution from myocarditis to dilated cardiomyopathy. Explanted heart of 28 patients, who underwent heart transplantation were screened for EV, AdV3 and HHV6 sequences in order to assess the incidence of cardiac viral infection that may be implicated in the pathogenesis of cardiomyopathy, and estimate viral distribution in the myocardium. Viral sequences were extracted from five different regions of the hearts. Nested PCR was used to amplify conservative regions of AdV3, HHV6 and EVs. Histological examination was performed on routinely processed myocardial samples. AdV3 was verified in one fourth of the patients. ADV3 and HHV6 sequences coexisted in one case with inflammatory cardiomyopathy. Some patients had more than one positive area of their heart. AdV3 positive right ventricular samples were double in amount compared to the left ones. None of the patients had positive result for EV. This is the first occasion to identify AdV3 (a mainly respiratory infective virus) sequence in explanted hearts of cardiomyopathy patients. Though the clinical importance of our results is still unclear, AdV3 could be a new member of the viral group with possible pathogenic effect on the myocardium. Regional distribution of viral sequence location confirmed that the right ventricular wall as a biopsy sampling site might be adequate for endomyocardial biopsy pro diagnostic purposes.

Electron microscopic demonstration of Romhányi's aldehyde-bisulphite-toluidine blue (ABT)-topo-optical staining reaction.

Romhányi's aldehyde-bisulphite-toluidine blue (ABT) topo-optical reaction was checked electron microscopically. The reaction product was found localized on the sarcolemma membrane and intercalated disc. The findings suggest that the topo-optical ABT-reaction is suitable for molecular analysis. In addition to confirming the electron microscopic suitability of the ABT-reaction it has also offered ultrastructural evidence for the reliability of the topo-optical method.

[Molecular biological virus identification in dilated cardiomyopathy]. / Víruskimutatás molekuláris biológiai vizsgálattal cardiomyopathiás betegek szívizommintáiból.

UNLABELLED: Enteroviruses have been considered to be the most common cause of acute myocarditis and possible consequence of dilated cardiomyopathy. Some publications shed light to the role of other viruses in this disease as well. Our molecular investigation has demonstrated that adeno- and herpes viruses might also frequently occur in dilated cardiomyopathy. AIM: The aim of our study was to screen virus genomes in heart tissues from heart-transplanted patients to prove their possible role in the pathogenesis of dilated cardiomyopathy. METHODS: DNA and RNA were isolated from five regions of the heart muscle. Amplification for Adenovirus Type 3, Human Herpes Virus Type 6 and Enterovirus genomes were performed by nested-Polymerase Chain Reaction. Finally the virus-positive samples were direct sequenced. RESULTS: In 2 patients Adenovirus Type 3 and in 1 patient both Adenovirus Type 3 and Human Herpes Virus Type 6 were detected. No enteroviruses were found in any heart tissue. CONCLUSIONS: In our study the adenovirus genome was found to be the most frequent virus genome in explanted heart tissues. The identified viral sequences proved previous viral infection, which could have played a role in the development of dilated cardiomyopathy. Detection of different viruses in the myocardium by molecular biological examinations might contribute to adequate treatment of these patients.

[Proteoglycans in the liver]. / Proteoglikánok a májban.

Proteoglycans are macromolecules formed by a protein core to which sugar chains are covalently attached. They are present on the cell surface and in the ECM of living things. In normal liver syndecan-1 is the dominant transmembrane proteoglycan, trace amounts of ECM proteoglycans are in the stromal components. The amounts of proteoglycans we studied increase in liver cirrhosis. In liver cancer abnormal localization of syndecan-1 and stroma rich in agrin was characteristic. The core proteins as well as the sugar chains of proteoglycans interact with and modulate the effect of regulatory factors. This implies that structural alterations of proteoglycans contribute to the development of malignant phenotype. Heparan sulfate chains of liver cancer are undersulfated with decreased or altered biological activity. Their binding capacity for transcription factor decreases, and they do not inhibit topoisomerase I enzyme. Truncated form of syndecan-1 lacking the extracellular domain of the molecule induces differentiation of hepatoma cell line and inhibits the shedding of syndecan-1. This phenomenon calls attention to the importance of syndecan-1 shedding in the regulation of cell behavior.