Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells.

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

ß-Amyloid Orchestrates Factor XII and Platelet Activation Leading to Endothelial Dysfunction and Abnormal Fibrinolysis in Alzheimer Disease.

Alzheimer disease (AD) is the most common form of dementia in humans. However, to date, the cause of sporadic AD (SAD), which is the most frequent form, is still unknown. Although it has not been possible to determine the origin of this disease, the amyloid hypothesis is one of the most accepted to explain the etiology of AD. This hypothesis proposes that the pathogenesis of AD is derived from the toxic effect produced by the amyloid-ß (Aß) peptide in the brain parenchyma, but it does not make clear how Aß is capable of producing such damage. Furthermore, it has been observed that SAD is accompanied by disruptions in the vascular system, such as damage to the blood-brain barrier. This facilitates the transfer of some systemic proteins, such as fibrinogen, to the brain parenchyma, where Aß is abundant. Therefore, this Aß interacts with fibrinogen, which favors the formation of clots resistant to fibrinolysis, inducing a risk of thrombosis and neuroinflammation. Notably, Aß is not only of neuronal origin; platelets also contribute to high Aß production in the circulation. The Aß present in circulation favors the activation of coagulation factor XII, which leads to the generation of thrombin and bradykinin. In addition to Aß-induced platelet activation, all these events favor the development of inflammatory processes that cause damage to the brain vasculature. This damage represents the beginning of the toxic effects of Aß, which supports the amyloid hypothesis. This review addresses the relationship between alterations in the vascular and hemostatic systems caused by Aß and how both alterations contribute to the progression of SAD.

Platelet activation as a trigger factor for inflammation and atherosclerosis.

Platelets, in addition to participating in atherosclerosis, play a very active role in the immune response of this disease since they have the ability to interact with various inflammatory cells, in addition to secreting cytokines, chemokines, growth factors, etc. The functions of platelets go beyond their interaction with the endothelium, as they participate in creating an inflammatory environment, which contributes to the loss of homeostasis. On the other hand, platelet-derived microparticles induce the activation of other platelets, of endothelial cells and in recruiting leukocytes. For all the above, platelets and the inflammatory environment can be considered as possible therapeutic targets to prevent the development of atherosclerosis and the events associated with it.

Las plaquetas, además de participar en la ateroesclerosis, desempeñan un papel muy activo en la respuesta inmunitaria de esta enfermedad, ya que tienen la capacidad de interaccionar con diversas células inflamatorias, además de secretar citocinas, quimiocinas, factores de crecimiento, etc. Las funciones de las plaquetas van más allá de su interacción con el endotelio, pues participan en crear un ambiente inflamatorio, lo que contribuye a la pérdida de la homeostasis. Por otra parte, las micropartículas derivadas de plaquetas inducen la activación de otras plaquetas y de células endoteliales, y el reclutamiento de leucocitos. Por todo lo anterior, las plaquetas y el ambiente inflamatorio pueden considerarse como posibles blancos terapéuticos para evitar el desarrollo de la ateroesclerosis y los eventos asociados a esta.

γδ T Cells Number, CD200, and Flt3 Expression Is Associated with Higher Progression Free Survival in Patients with Chronic Myeloid Leukemia.

BACKGROUND: Tumor immunoedition involves alterations in cells of immune system, which may play an important role in the immunosurveillance of patients with cancer diseases. AIM OF THE STUDY: To determine the association between the number of immune cells and the expression of surface markers in leukemic cells of patients with de novo CML who achieved molecular response. METHODS: A longitudinal study was conducted in 31 patients with de novo CML. Peripheral blood samples were obtained at diagnosis for quantification of immune cells and tumor cells expressing CD200, CD135, GpP, and Bcl-2. Results were compared with a group of 60 healthy donors. Lymphocyte subsets were analyzed during a 48 month follow-up period and molecular response to treatment was assessed simultaneously by QT-PCR. The group of patients with deep molecular response was compared with de novo CML patients; the cut-off value of cell count was determined by ROC analysis. Kaplan-Meier and Cox proportional hazard model were used to determine the significant association between the number of cells and progression-free survival. RESULTS: Differences in number of CD4, CD4Tregs, NK, γδT, monocytes, and pDC's, tumor-cells expressing CD200+, CD135+, GpP+, and Bcl-2+ were observed between patients and healthy donors. The number of γδT lymphocytes, CD200+, and CD135+ cells were associated with longer progression-free survival (p = 0.0112, p = 0.0012 and p = 0.0201 respectively). CONCLUSION: A γδT lymphocyte count <63 cel/uL, CD200+ <997 cel/uL, and CD135+ <23 317 cel/uL at diagnosis is associated with the maintenance of deep molecular response at 48 months in patients with de novo CML.

Cell proliferation and inhibition of apoptosis are related to c-Kit activation in leukaemic lymphoblasts.

Receptor tyrosine kinase (RTK) activity may contribute to carcinogenesis. The c-Kit receptor, a member of the RTK family, is expressed in immature haematopoietic system cells. Acute lymphoblastic leukaemia (ALL) presents incompletely differentiated lymphoblasts, and consequently, c-Kit expression can be detected in these cells. The BCR-ABL kinase, which is usually present in both ALL and chronic myeloid leukaemia, can trigger signalling pathways with neoplastic effects. However, a certain number of ALL patients and chronic myeloid leukaemia patients do not express this kinase, raising the question of which other proteins that intervene in signalling pathways may be involved in the development of these diseases. OBJECTIVES: To test whether c-Kit has proliferative effects and affects the inhibition of apoptosis of leukaemic lymphoblasts that do not express BCR-ABL. METHODS: We cultured RS4:11 lymphoblasts and analysed the expression and activation of c-Kit by immunofluorescence, and flow cytometry, evaluation of cell proliferation, apoptosis, cyclin D1 and Bak expression were carried out by flow cytometry; activation of AKT and survivin expression were tested by immunoblot. RESULTS: The c-Kit receptor was found to induce proliferation and to increase the expression of cyclin D1 via the PI3K/AKT/NF-kB signalling pathway. Additionally, the c-Kit/PI3K/AKT pathway increased the inhibition of apoptosis and survivin expression. Similarly, c-Kit was observed to reduce the expression of the pro-apoptotic Bak protein. CONCLUSION: These results suggest that, in leukaemic lymphoblasts, c-Kit triggers a signalling pathway with proliferative and anti-apoptotic effects; information to this effect has not yet been reported in the literature.

A novel ß-catenin signaling pathway activated by IL-1ß leads to the onset of epithelial-mesenchymal transition in breast cancer cells.

Interleukin 1ß has been associated with tumor development, invasiveness and metastasis in various types of cancer. However, the molecular mechanisms underlying this association have not been clearly elucidated. The present study is the first to show, in breast cancer cells, that an IL-1ß/IL-1RI/ß-catenin signaling pathway induces ß-catenin accumulation due to GSK3ß inactivation by Akt phosphorylation. Translocation to the nucleus of accumulated ß-catenin and formation of the TCF/Lef/ß-catenin complex induce sequential expression of c-MYC, CCDN1, SNAIL1 and MMP2, leading to up-regulation of proliferation, migration and invasion; all of the processes shown to be required, in cancerous cells, to initiate transition from a non-invading to an invasive phenotype.

Undifferentiated immunophenotypes and not expression of BCR-ABL can be associated in adult Mestizo Mexican patients with ALL.

INTRODUCTION: The BCR-ABL t(9;22)(q34;q11) translocation has been identified as a risk factor in de novo acute lymphoblastic leukemia (ALL), but there are other factors that may influence survival in patients not expressing this translocation. OBJECTIVE: To associate expression and non-expression of BCR-ABL with immunophenotype and other clinical features in adult patients with ALL from a Mexican mestizo population. MATERIAL AND METHODS; Peripheral blood samples from 35 adult patients with de novo ALL were used to detect BCR-ABL by reverse transcriptase polymerase chain reaction (RT-PCR) as well as immunophenotype by flow cytometry. RESULTS: In the group of BCR-ABL negative patients (74.28%) two subgroups were identified with the immature immunophenotypes CD34+/CD33+ and/or CD13+, and CD10-/CD34+. In the group of BCR-ABL positive patients (25.72%) leukemic blast cells with a more differentiated immunophenotype compared to the BCR-ABL negative group were found. As regards clinical and biological characteristics, we found survival in months to be very similar and a tendency to high initial leukocyte counts in both groups. CONCLUSIONS: This is the first study conducted on a Mexican mestizo population to report that BCR-ABL negative patients can present a high frequency of undifferentiated immunophenotypes and must therefore be considered as vulnerable as BCR-ABL positive patients.

Src, Akt, NF-κB, BCL-2 and c-IAP1 may be involved in an anti-apoptotic effect in patients with BCR-ABL positive and BCR-ABL negative acute lymphoblastic leukemia.

BCR-ABL kinase has been observed to be potentially related to leukemic cell development. Adult patients with acute lymphoblastic leukemia (ALL) were evaluated to determine whether presence/absence of BCR-ABL induced differences in activation of Src, PI3K/Akt and NF-κB or in the expression of anti-apoptotic proteins such as BCL-2 and c-IAP1. BCR-ABL positive patients showed a significantly higher activation of Src and Akt compared with BCR-ABL negative patients and healthy donors. BCR-ABL negative patients also showed a significant activation of Src and low levels of Akt activation compared with healthy donors. Both patient groups had increased NF-κB activation and overexpression of BCL-2 and c-IAP1. This is the first study to evaluate concurrently in ALL patients presence/absence of BCR-ABL in relation to activation of Src, Akt and NF-κB and the expression of anti-apoptotic proteins. Results suggest that these proteins may be involved in an anti-apoptotic signaling pathway.

Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain.

Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445 kb which could encode a protein of 16.88 kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15 kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.

Heat shock protein 60 from Klebsiella pneumoniae protects mononuclear cells from apoptotic cell death induced by dexamethasone.

AIMS: Bacterial heat shock proteins can have anti-apoptotic effects on human cells. We investigated whether enterobacterial HSP60 can protect peripheral blood mononuclear cells (PBMC) from DXM-induced apoptosis and if this effect requires cytoskeleton participation. MAIN METHODS: Anti-apoptotic effect from enterobacterial HSP60 was analyzed by adding these proteins to peripheral mononuclear cells cultures before DXM induction. Percentage of apoptotic cells was determined by SubG0 peak and TUNEL techniques in a flow cytometer. KEY FINDINGS: Our results showed significant protective effect of HSP60 Klebsiella pneumoniae and E. coli, in the DXM-induced apoptosis in PBMC. Similar results were obtained with recombinant human HSP60. The same protective effect of proteins was observed in CD4+ and CD8 + T cell subpopulations. To analyze if enterobacterial HSP60 need internalization to have the anti-apoptotic effect, we used cytoskeleton inhibitors such as: nocodazole, cytochalasin D and amiloride, the three inhibitors significantly affected the protective role of HSP60 in apoptosis induced with DXM. Results suggest that the protective effect of HSP60 K. pneumoniae and E. coli requires the participation of contractile structures for the internalization of this protein by the cells, we suggest that the internalization of enterobacterial HSP60 could be carry out by macropinocytosis. SIGNIFICANCE: We report for the first time that K. pneumoniae and E. coli HSP60 have protective effect in the apoptosis induced with DXM in PBMC from healthy subjects and that this effect requires the internalization of the protein with active participation of the cytoskeleton.

Mechanisms of cell volume regulation in hypo-osmolality.

Cells respond to a condition of hypo-osmolality by rapid swelling followed by an adaptive response that tends to recover the normal cell volume despite the persistence of the hypo-osmotic condition. This is an active process accomplished by the extrusion of intracellular osmolytes, essentially K+, Cl-, and small organic molecules. This regulatory process operates through a chain of events that essentially consists of a sensor or sensing mechanism to detect changes in cell volume, a signaling cascade to amplify the sensing signal and orient it to activate pathways for osmolyte extrusion, and a memory of the original cell volume, which sets the timing for inactivation of the volume-regulatory process. This article presents a brief overview of recent progress in these different aspects of the volume-regulatory process, including (1) the mechanisms and/or candidate molecules serving the role of volume sensors, (2) the osmosignaling network and the interplay and hierarchy of the different elements in this chain, and (3) the nature and properties of the osmolyte extrusion pathways. Emphasis is placed on some of the main unsolved questions concerning different aspects of the volume-regulatory process. Recent findings regarding the effect of hypo-osmolality on synaptic function are briefly discussed in terms of the possible molecular basis for the neurologic symptoms induced by hyponatremia.

Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons.

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Osmolytes and mechanisms involved in regulatory volume decrease under conditions of sudden or gradual osmolarity decrease.

A decrease in external osmolarity results in cell swelling and the immediate activation of a mechanism to restore cell volume, known as regulatory volume decrease (RVD). When exposed to a gradual osmolarity decrease (GODE), some cells do not swell. This reflects the operation of an active regulatory process known as isovolumetric regulation (IVR). The mechanisms underlying IVR appear similar to those activated during RVD, namely the extrusion of K+, Cl-, amino acids, and other organic molecules. A previous study has documented IVR in cerebellar granule neurons, parallel to an early efflux of taurine and Cl-, whereas K+ efflux is delayed. In this work we briefly review the importance of amino acids in the mechanisms of cell volume control in the brain, with emphasis on IVR. We also present experiments showing the response to GODE in cerebellar astrocytes. The currents activated during GODE, recorded in the whole-cell configuration of the patch clamp technique, indicate the early activation of an anion current, followed by a more delayed cation current. A correlation between the time course of amino acid efflux during GODE and the occurrence or not of IVR in various cell types, suggest the importance of these osmolytes in the volume regulatory process in this model.

Epidermal growth factor receptor is activated by hyposmolarity and is an early signal modulating osmolyte efflux pathways in Swiss 3T3 fibroblasts.

Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically induced taurine release, as suggested by the latter's reduction when EGFR phosphorylation was blocked by 50 microM AG213 or AG112 and, conversely, its potentiation by EGF (200 ng/ml). The relationship between hyposmotically induced taurine efflux and reduced osmolarity showed saturable kinetics, following a sigmoidal function. EGF shifted the relationship to the left, implying an increase in sensitivity to hyposmolarity. EGF increased taurine efflux only marginally under isosmotic conditions. EGF and agglutinins also potentiated the hyposmotically induced release of 86Rb but, in contrast to taurine, the efflux was unaffected by EGFR inhibition. EGF and agglutinins markedly increased 86Rb release under isosmotic conditions. The EGF-evoked isosmotic 86Rb release, together with the hyposmotic efflux, accounted fully for the observed potentiation by EGF, raising the possibility of an overlapping of these two effects, rather than a true potentiation. A link between EGFR, phosphatidylinositide-3-kinase (PI3K) and hyposmotically induced taurine (but not 86Rb) release is suggested by the increase in PI3K activity elicited by hyposmolarity, which was fully prevented by EGFR inhibition, and by a marked reduction of hyposmotically induced taurine (but not 86Rb) release, by wortmannin. The present findings, together with results showing EGF activation of osmosensitive Cl- fluxes implicate EGFR as an important modulator of osmolyte efflux pathways.

Potentiation of the osmosensitive taurine release and cell volume regulation by cytosolic Ca2+ rise in cultured cerebellar astrocytes.

Hyposmolarity (-30%) in cultured cerebellar astrocytes raised cytosolic Ca2+ concentration ([Ca2+]i) from 160 to 400 nM and activated the osmosensitive taurine release (OTR) pathway. Although OTR is essentially [Ca2+]i-independent, further increase in [Ca2+]i by ionomycin strongly enhanced OTR, with a more robust effect at low and mild osmolarity reductions. Ionomycin did not affect isosmotic taurine efflux. OTR was decreased by tyrphostin A25 and increased by ortho-vanadate, suggesting a modulation by tyrosine kinase or phosphorylation state. Inhibition of phosphatidylinositol-3-kinase activity by wortmannin markedly decreased OTR and the ionomycin increase. Conversely, OTR and the ionomycin effect were independent of ERK1/ERK2 activation. OTR and its potentiation by ionomycin differed in their sensitivity to CaM and CaMK blockers and in the requirement of an intact cytoskeleton for the ionomycin effect, but not for normal OTR. Changes in the actin cytoskeleton organization elicited by hyposmolarity were not observed in ionomycin-treated cells, which may permit the operation of CaM/CaMK pathways involved in the OTR potentiation by [Ca2+]i rise. OTR potentiation by [Ca2+]i requires the previous or simultaneous activation/operation of the taurine release mechanism and is not modifying its set point, but rather increasing the effectiveness of the pathway, resulting in a more efficient volume regulation. This may have a beneficial effect in pathological situations with concurrent swelling and [Ca2+]i elevation in astrocytes.

Tyrosine kinases and amino acid efflux under hyposmotic and ischaemic conditions in the chicken retina.

The chicken retina was exposed to 20% hyposmotic or ischaemia-like (54 mM KCl and 1 mM ouabain) conditions and changes in cell volume, amino acid release and activation of protein tyrosine kinases measured. To investigate possible connection between these cellular events, the effect of tyrosine kinase blockers on (3)H-taurine, (3)H-GABA and (3)H- D-aspartate (as a tracer for glutamate) efflux was examined. Both hyposmotic and ischaemic conditions increased phosphorylation of the tyrosine kinase p125 focal adhesion kinase (p125(FAK)) and the mitogen-activated protein kinase-p38 (MAPK-p38), but not of the extracellular-signal-related kinases-1/2 (ERK1/ERK2), and markedly activated the tyrosine kinase target enzyme phosphatidylinositide 3-kinase (PI3K). Hyposmolarity and ischaemia both led to rapid retinal swelling followed by active volume recovery of 84% (hyposmolarity) and 40% (ischaemia), together with rapid release of taurine, GABA and D-aspartate. Taurine and GABA efflux under both conditions was reduced markedly by tyrosine kinase and PI3K blockers (50 microM tyrphostin A23, 50 microM genistein, 100 nM wortmannin, 25 microM LY294002) and was decreased by 85% when ischaemia-induced swelling was prevented. About 65% of D-aspartate efflux occurred irrespective of swelling in ischaemia and was either less sensitive (hyposmotic) or largely resistant (ischaemia) to the blockers. These results suggest that in ischaemia, GABA and taurine react primarily to swelling with a typical osmolyte response, while glutamate differs in its release mechanisms under both hyposmotic and ischaemic conditions. These findings suggest new strategies for evaluating the contribution of swelling to excitotoxicity in ischaemia.

Myosin I interactions with actin filaments and trans-golgi-derived vesicles in MDCK cell monolayers

In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma memebrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of memebrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association ot the actin binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with tran-Golgi-derived vesicle. Modifications of the ring were induced subjecting the cells to external Ca²+ switch), or by treatment with drugs known to depolymerize actin filament (cytochalasin D, CD). The distribution of myosin I and actin, both in intact cells and in cellular fractions, was monitored using heterlogous cross-reacting antibodies and phalloidin. The authors identified an isoform of myosin I of approximately 110-125 KDa, homologus to myosin IB of Acanthamoeba, a fraction of wich colocalized with the peripheral actin ring. The association seems transient as, once the ring retracted as result of Ca²+ depletion, or became disroganized by CD, myosin not longer colocalized with the actin fibers but appeared dispersed in the cytoplasm. Furthermore, a signficant fraction of the total myosin I in the cell was associated to Golgi-derived vesicles which could also associate in vitro with actin filaments. The authors' data support, then, the participation of myosin I, in association with actin filaments, in vesicle translocation to and from the cell membrane as proposed for natural epithelia, and provide a further insigh into the structural organization that maintains epithelial cell polatiry in cultured monolayers