Violacein improves vemurafenib response in melanoma spheroids.

Chemotherapy resistance is one of the main challenges in melanoma treatment. Violacein, a natural pigment produced by Chromobacterium violaceum, induces apoptosis in a variety of tumours, including melanoma. Here, we used BRAF-mutated melanoma spheroids to test the potential of violacein as a sensitizer of cellular viability and levels of the proteins p62 and fatty acid synthase (FASN). Importantly, violacein in combination with vemurafenib (ViVe) was able to interfere with spheroid survival at subtoxic concentrations. The results demonstrated that the ViVe protocol triggered cell death assessed by calcein and ethidium homodimer dyes. Accordingly, melanoma cells in 2D systems also showed a higher apoptosis rate when treated with ViVe. In the current study, we show evidence that ViVe downregulates crucial mediators like FASN, which partially explains how it acts as a sensitizer and ultimately improves the effectiveness of vemurafenib against melanoma cells.

A colloidal hydrogel-based drug delivery system overcomes the limitation of combining bisphosphonates with bioactive glasses: in vitro evidence of a potential selective bone cancer treatment allied with bone regeneration.

Bisphosphonates are a class of drugs that induce bone cancer cell death and favor bone regeneration, making them suitable for bone cancer treatment. However, when combined with bioactive glasses to enhance bone regeneration, a chemical bond between biphosphonates and the glass surface inactivates their mechanism of action. A new colloidal hydrogel-based drug delivery system could overcome that limitation once bisphosphonates, such as zoledronic acid (ZA), are incorporated into hydrogel micelles, avoiding their interaction with the glass surface. In this work, we proposed formulations based on a poloxamer 407 thermo-responsive hydrogel matrix containing holmium-doped bioactive glass nanoparticles and different concentrations (0.05 and 5 mg/mL) of ZA. We characterized the influence of the glass and the ZA on the hydrogel properties. In addition, a drug concentration screening was performed, and biological characterizations evaluated the best result. The biological characterization consisted of evaluating cytotoxicity and in vitro bone regeneration ability through cell migration and quantification of genes related to osteogeneses through RT-PCR. The results suggest that the addition of glasses and ZA to the poloxamer did not significantly influence the sol-gel transition of the hydrogels (around 13 °C) regardless of the ZA content. However, the ZA at high concentration (PL-ZA100) decreased the enthalpy of gel formation from 68 to 43 kJ.mol-1 when compared with the pure hydrogel formulation (PL), suggesting a water structurer role of ZA, which is withdrawn when glass particles are added to the system (PL-BG5Ho-ZA100). Solid-state 31P nuclear resonance spectroscopy results showed that part of the ZA is chemically bonded to the glass surface, which explains the withdrawal in the water structurer role of ZA when the glasses were incorporated into the hydrogel. Besides, based on the drug release results, we proposed a model where part of the ZA is "free," encapsulated in the hydrogel matrix, while another part of the ZA is bonded to the glass surface. Finally, considering the in vitro results and our proposed model, the ratio between "free" and "bonded" ZA in our drug delivery systems showed in vitro evidence of a cancer treatment that selectively kills osteosarcoma cells while still favoring an osteogenic microenvironment. By overcoming the limitation of combining bisphosphonates with bioactive glasses, hydrogel-based drug delivery systems can be a solution for the development of new formulations proposed for bone cancer treatment in conjunction with bone regeneration.

Expression of thimet oligopeptidase (THOP) modulated by oxidative stress in human multidrug resistant (MDR) leukemia cells.

Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after H2O2 treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.

Bioactive Glasses as Carriers of Cancer-Targeted Drugs: Challenges and Opportunities in Bone Cancer Treatment.

The treatment of bone cancer involves tumor resection followed by bone reconstruction of the defect caused by the tumor using biomaterials. Additionally, post-surgery protocols cover chemotherapy, radiotherapy, or drug administration, which are employed as adjuvant treatments to prevent tumor recurrence. In this work, we reviewed new strategies for bone cancer treatment based on bioactive glasses as carriers of cancer-targeted and other drugs that are intended for bone regeneration in conjunction with adjuvant treatments. Drugs used in combination with bioactive glasses can be classified into cancer-target, osteoclast-target, and new therapies (such as gene delivery and bioinorganic). Microparticulated, nanoparticulated, or mesoporous bioactive glasses have been used as drug-delivery systems. Additionally, surface modification through functionalization or the production of composites based on polymers and hydrogels has been employed to improve drug-release kinetics. Overall, although different drugs and drug delivery systems have been developed, there is still room for new studies involving kinase inhibitors or antibody-conjugated drugs, as these drugs have been poorly explored in combination with bioactive glasses.

A canthin-6-one derivative induces cell death by apoptosis/necroptosis-like with DNA damage in acute myeloid cells.

Natural products have long been considered a relevant source of new antitumor agents. Despite advances in the treatment of younger patients with acute myeloid leukemia (AML), the prognosis of elderly patients remains poor, with a high frequency of relapse. The cytotoxicity of canthin-6-one alkaloids has been extensively studied in different cell types, including leukemic strains. Among the canthin-6-one analogs tested, 10-methoxycanthin-6-one (Mtx-C) showed the highest cytotoxicity in the malignant AML cells Kasumi-1 and KG-1. Thus, we evaluated the cytotoxicity and cell death mechanisms related to Mtx-C using the EC50 (80 µM for Kasumi-1 and 36 µM for KG-1) treatment for 24 h. Our results identify reactive oxygen species production, mitochondrial depolarization, annexin V-FITC/7-AAD double staining, caspase cleave and upregulation of mitochondria-dependent apoptosis proteins (Bax, Bim, Bik, Puma and phosphorylation of p53) for both cell lineages. However, downregulation of Bcl-2 and the simultaneous execution of the apoptotic and necroptotic programs associated with the phosphorylation of the proteins receptor-interacting serine/threonine-protein kinase 3 and mixed lineage kinase domain-like pseudokinase occurred only in Kasumi-1 cells. About the lasted events, Kasumi-1 cell death was inhibited by pharmacological agents such as Zvad-FMK and necrostatin-1. The underlying molecular mechanisms of Mtx-C still include participation in the DNA damage and stress-signaling pathways involving p38 and c-Jun N-terminal mitogen-activated protein kinases and interaction with DNA. Thus, Mtx-C represents a promising tool for the development of new antileukemic molecules.

The Glypican proteoglycans show intrinsic interactions with Wnt-3a in human prostate cancer cells that are not always associated with cascade activation.

BACKGROUND: Prostate cancer occurs through multiple steps until advanced metastasis. Signaling pathways studies can result in the identification of targets to interrupt cancer progression. Glypicans are cell surface proteoglycans linked to the membrane through glycosylphosphatidylinositol. Their interaction with specific ligands has been reported to trigger diverse signaling, including Wnt. In this study, prostate cancer cell lines PC-3, DU-145, and LNCaP were compared to normal prostate RWPE-1 cell line to investigate glypican family members and the activation of the Wnt signaling pathway. RESULTS: Glypican-1 (GPC1) was highly expressed in all the examined cell lines, except for LNCaP, which expressed glypican-5 (GPC5). The subcellular localization of GPC1 was detected on the cell surface of RWPE-1, PC-3, and DU-145 cell lines, while GPC5 suggested cytoplasm localization in LNCaP cells. Besides glypican, flow cytometry analysis in these prostate cell lines confirmed the expression of Wnt-3a and unphosphorylated ß-catenin. The co-immunoprecipitation assay revealed increased levels of binding between Wnt-3a and glypicans in cancer cells, suggesting a relationship between these proteoglycans in this pathway. A marked increase in nuclear ß-catenin was observed in tumor cells. However, only PC-3 cells demonstrated activation of canonical Wnt signaling, according to the TOPFLASH assay. CONCLUSIONS: GPC1 was the majorly expressed gene in all the studied cell lines, except for LNCaP, which expressed GPC5. We assessed by co-immunoprecipitation that these GPCs could interact with Wnt-3a. However, even though nuclear ß-catenin was found increased in the prostate cancer cells (i.e., PC-3, DU-145 and LNCaP), activation of Wnt pathway was only found in PC-3 cells. In these PC-3 cells, GPC1 and Wnt-3a revealed high levels of colocalization, as assessed by confocal microscopy studies. This suggests a localization at the cellular surface, where Frizzled receptor is required for downstream activation. The interaction of Wnt-3a with GPCs in DU-145 and LNCaP cells, which occurs in absence of Wnt signaling activation, requires further studies. Once non-TCF-LEF proteins can also bind ß-catenin, another signaling pathway may be involved in these cells with regulatory function.

Heparanase modulation by Wingless/INT (Wnt).

Heparanase is an endo-beta-glucuronidase, the only enzyme in mammals capable of cleaving heparan sulfate/heparin chains from proteoglycans. The oligosaccharides generated by heparanase present extensive biological functions since such oligosaccharides interact with adhesion molecules, growth factors, angiogenic factors and cytokines, modulating cell proliferation, migration, inflammation, and carcinogenesis. However, the regulation of heparanase activity is not fully understood. It is known that heparanase is synthesized as an inactive 65 kDa isoform and that post-translation processing forms an active 50 kDa enzyme. In the present study, we are interested in investigating whether heparanase is regulated by its own substrate as observed with many other enzymes. Wild-type Chinese hamster (Cricetulus griséus) ovary cells (CHO-K1) were treated with different doses of heparin. Heparanase expression was analyzed by Real-time PCR and flow cytometry. Also, heparanase activity was measured. The heparanase activity assay was performed using a coated plate with biotinylated heparan sulfate. In the present assay, a competitive heparin inhibition scenario was set aside. Exogenous heparin trigged a cell signaling pathway that increased heparanase mRNA and protein levels. The Wnt/beta-catenin pathway, judged by TCF-driven luciferase activity, seems to be involved to enhance heparanase profile during treatment with exogenous heparin. Lithium chloride treatment, an activator of the Wnt/beta-catenin pathway, confirmed such mechanism of transduction in vivo using zebrafish embryos and in vitro using CHO-K1 cells. Taken together the results suggest that heparin modulates heparanase expression by Wnt/beta-catenin.

Divergent effects of Wnt5b on IL-3- and GM-CSF-induced myeloid differentiation.

The multiple specialized cell types of the hematopoietic system originate from differentiation of hematopoietic stem cells and progenitors (HSPC), which can generate both lymphoid and myeloid lineages. The myeloid lineage is preferentially maintained during ageing, but the mechanisms that contribute to this process are incompletely understood. Here, we studied the roles of Wnt5a and Wnt5b, ligands that have previously been linked to hematopoietic stem cell ageing and that are abundantly expressed by both hematopoietic progenitors and bone-marrow derived niche cells. Whereas Wnt5a had no major effects on primitive cell differentiation, Wnt5b had profound and divergent effects on cytokine-induced myeloid differentiation. Remarkably, while IL-3-mediated myeloid differentiation was largely repressed by Wnt5b, GM-CSF-induced myeloid differentiation was augmented. Furthermore, in the presence of IL-3, Wnt5b enhanced HSPC self-renewal, whereas in the presence of GM-CSF, Wnt5b accelerated differentiation, leading to progenitor cell exhaustion. Our results highlight discrepancies between IL-3 and GM-CSF, and reveal novel effects of Wnt5b on the hematopoietic system.

Crosstalk between tumor cells and lymphocytes modulates heparanase expression.

BACKGROUND: Heparanase (HPSE) is an endo-beta-glucuronidase that degrades heparan sulfate (HS) chains on proteoglycans. The oligosaccharides generated by HPSE promote angiogenesis, tumor growth and metastasis. Heparanase-2 (HPSE2), a close homolog of HPSE, does not exhibit catalytic activity. Previous studies have demonstrated that serum or plasma from breast cancer patients showed increased expression of both heparanases in circulating lymphocytes. The aim of this study was to better understand the mechanisms involved in the upregulation of heparanases in circulating lymphocytes. METHODS: Lymphocytes collected from healthy women were incubated in the presence of MCF-7 breast cancer cells (co-culture) to stimulate HPSE and HPSE2 overexpression. The protein level of heparanases was evaluated by immunocytochemistry, while mRNA expression was determined by quantitative RT-PCR. RESULTS: The medium obtained from co-culture of MCF-7 cells and circulating lymphocytes stimulated the expression of HPSE and HPSE2. Previous treatment of the co-culture medium with an anti-heparan sulfate proteoglycan antibody or heparitinase II inhibited the upregulation of heparanases in circulating lymphocytes. The addition of exogenous heparan sulfate (HS) enhanced the expression of both heparanases. Moreover, the co-cultured cells, as well as MCF-7 cells, secreted a higher number of exosomes expressing an increased level of HS compared to that of the exosomes secreted by circulating lymphocytes from women who were not affected by cancer. CONCLUSIONS: The results revealed that HS is likely responsible for mediating the expression of heparanases in circulating lymphocytes. HS secreted by tumor cells might be carried by exosome particles, confirming the key role of tumor cells, as well as secreted HS, in upregulating the expression of heparanases, suggesting a possible mechanism of crosstalk between tumor cells and circulating lymphocytes.

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines.

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L-cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G 0 and G 2 phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.

Interaction of violacein in models for cellular membranes: Regulation of the interaction by the lipid composition at the air-water interface.

Some biological properties of violacein are believed to be associated with their interactions with lipid surfaces, encouraging research on the identification of membrane sites capable of drug binding. In this study, we investigated the interaction of violacein with cell membrane models represented by Langmuir monolayers of selected lipids: one representing healthy cellular membranes: dipalmitoylphosphatidylcholine, DPPC, and the other one representing tumorigenic cellular membranes, dipalmitoylphosphatidylserine, DPPS. It is shown that even small amounts of the compound affect the surface pressure-area isotherms as well as the surface vibrational spectra of the lipid monolayers, which points to a significant interaction. Such effects depend on the electrical charge of the monolayer-forming molecules, with the drug activity being particularly distinctive for negatively charged lipids in relation to zwitterionic lipids. Morphological analysis also suggests that violacein at the air-water interface is homogenized when incorporated in both lipids. Although the interaction of violacein with the lipids affects viscoelastic and structural properties of the Langmuir monolayer, it is not present permeability through lipid bilayers, as investigated using liposomes. These results therefore may have important implications in understanding how violacein acts on specific sites of the cellular membrane, and evidence the fact that the lipid composition of the monolayer modulates the interaction with the lipophilic drug.

Action and function of Chromobacterium violaceum in health and disease: Violacein as a promising metabolite to counteract gastroenterological diseases.

Chromobacterium violaceum is a Gram negative, ß-proteobacterium found in the microbiota of tropical and subtropical environments. Although considered an opportunistic pathogen, infection rapidly progress to fatal sepsis, with metastatic abscesses. It is noteworthy the multidrug resistant phenotype of C. violaceum and the possibility of relapse. Recently, an influence of global climate in the incidence of cases beyond the previous areas has been observed. Furthermore, chronic granulomatous disease has been considered a risk factor to infection. Despite the increase in C. violaceum infection incidence and high mortality, most clinicians are not familiar with it. This review pointed out important features of this life threatening microorganism, including its pathogenicity, mechanistic aspects, genetic and drug resistance associated factors, and the clinical association with chronic granulomatous disease. In addition, its main metabolite violacein may be a promising agent to counteract gastroenterological diseases, such as colorectal cancer and inflammatory gastric lesions.

Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

BACKGROUND: All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. METHODS: Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. RESULTS: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. CONCLUSION: Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

Cells involved in extracellular matrix remodeling after acute myocardial infarction.

OBJECTIVE: Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. METHODS: Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. RESULTS: There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. CONCLUSION: The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct.

Cells involved in extracellular matrix remodeling after acute myocardial infarction / Células envolvidas no remodelamento da matriz extracelular após infarto agudo do miocárdio

Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. .

Objetivo Avaliar os efeitos da transferência gênica do VEGF165 no processo de remodelamento da matriz extracelular após infarto agudo do miocárdio. Métodos Ratos Wistar foram submetidos ao infarto do miocárdio por ligação da artéria coronária descendente esquerda, e a fração de ejeção de ventrículo esquerdo foi utilizada para classificar os infartos em grandes e pequenos. Os animais foram divididos em grupos de dez animais, de acordo com o tamanho do infarto (grande ou pequeno), e receberam ou não tratamento com o VEGF165. A avaliação dos diferentes marcadores foi realizada por imuno-histoquímica e quantificação digital. Os anticorpos primários utilizados foram antifibronectina, antivimentina, anti- CD44, anti-E-caderina, anti-CD24, anti-alfa-1-actina e anti-PCNA. Os resultados foram representados como média e erro padrão, e analisados por ANOVA, sendo considerado estatisticamente significativo se p≤0,05. Resultados Houve aumento significativo da expressão de marcadores de células indiferenciadas, como fibronectina (proteína presente na matriz extracelular) e CD44 (glicoproteína presente nas células endoteliais). Entretanto, houve diminuição da expressão de vimentina e PCNA, indicando possível diminuição do processo de proliferação celular após o tratamento com VEGF165. Os marcadores de células diferenciadas, E-caderina (proteína de adesão entre as células do miocárdio), CD24 (proteína presente nos vasos sanguíneos) e alfa-1-actina (marcador especifico de miócitos) também apresentaram maior expressão nos grupos submetidos à terapia gênica, comparativamente com o grupo não tratado. O valor obtido pela relação entre alfa-1-actina e vimentina foi aproximadamente três vezes maior nos grupos tratados com VEGF165, indicando maior diferenciação tecidual. Conclusão O papel dos miócitos se mostrou importante no processo de remodelamento tecidual, confirmando que o VEGF165 parece conferir um efeito protetor no tratamento do infarto agudo do miocárdio. .

Hematopoietic modulators as potential agents for the treatment of leukemia.

Leukemias are the most common malignancy of childhood and have the highest mortality among aging people. Leukemias are a group of blood disorders characterized by an accumulation of leukemic cells in the peripheral blood of patients as a result of disturbances in proliferation and differentiation. Refractory leukemia remains the most common therapeutic challenge. In recent years, the presence of a cancer stem cell population in leukemias has been proposed as a cause for the refractory phenomenon. Insights into the cellular and molecular features of leukemia led to a new point of view in the choice of novel therapeutic agents. New agents for the treatment of this disease should selectively target leukemia stem cells or exhibit higher cytotoxic effects in cancer cells than in normal cells. A special interest is focused on anticancer agents from biological and natural sources that can be used in the treatment of leukemia. This review discusses the characteristics of some of these potential new agents.

Defining the molecular basis of tumor metabolism: a continuing challenge since Warburg's discovery.

Cancer cells are the product of genetic disorders that alter crucial intracellular signaling pathways associated with the regulation of cell survival, proliferation, differentiation and death mechanisms. The role of oncogene activation and tumor suppressor inhibition in the onset of cancer is well established. Traditional antitumor therapies target specific molecules, the action/expression of which is altered in cancer cells. However, since the physiology of normal cells involves the same signaling pathways that are disturbed in cancer cells, targeted therapies have to deal with side effects and multidrug resistance, the main causes of therapy failure. Since the pioneering work of Otto Warburg, over 80 years ago, the subversion of normal metabolism displayed by cancer cells has been highlighted by many studies. Recently, the study of tumor metabolism has received much attention because metabolic transformation is a crucial cancer hallmark and a direct consequence of disturbances in the activities of oncogenes and tumor suppressors. In this review we discuss tumor metabolism from the molecular perspective of oncogenes, tumor suppressors and protein signaling pathways relevant to metabolic transformation and tumorigenesis. We also identify the principal unanswered questions surrounding this issue and the attempts to relate these to their potential for future cancer treatment. As will be made clear, tumor metabolism is still only partly understood and the metabolic aspects of transformation constitute a major challenge for science. Nevertheless, cancer metabolism can be exploited to devise novel avenues for the rational treatment of this disease.

Profiling the changes in signaling pathways in ascorbic acid/ß-glycerophosphate-induced osteoblastic differentiation.

Despite numerous reports on the ability of ascorbic acid and ß-glycerophosphate (AA/ß-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/ß-GP-induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/ß-GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR-1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood.

Riboflavin and photoproducts in MC3T3-E1 differentiation.

Photoderivatives of riboflavin can modulate the proliferation and survival of cancer cells. In this work, we examined the influence of riboflavin and photoderivatives on osteoblast differentiation induced by ascorbic acid and ß-glycerophosphate. These compounds decreased the osteoblast proliferation, increased the alkaline phosphatase activity, promoted a reduction in matrix metalloproteinase-2 activity and the decreased in the OPG/RANKL ratio. The effects of flavins on osteoblasts were unrelated to the antioxidant activity of these compounds. The biological activity of osteogenic medium containing riboflavin and its photoderivatives involved the activation of different signaling pathways (AKT, FAK, CaMKII), caspases-3, -8 and -9, and up-regulation of the expression and/or stabilization of osteoblastic transcription factors (Runx2 and ß-catenin). These findings suggest a potential use of flavins as adjuvants to improve bone metabolism.

Heterozygosis for CYP21A2 mutation considered as 21-hydroxylase deficiency in neonatal screening / Heterozigose para mutação no gene CYP21A2 considerada como deficiência de 21-hidroxilase na triagem neonatal

Steroid 21-hydroxylase deficiency (21-OHD) accounts for more than 90 percent of congenital adrenal hyperplasia. CAH newborn screening, in general, is based on 17-hydroxyprogesterone dosage (17-OHP), however it is complicated by the fact that healthy preterm infants have high levels of 17-OHP resulting in false positive cases. We report on molecular features of a boy born pre-term (GA = 30 weeks; weight = 1,390 g) with elevated levels of 17-OHP (91.2 nmol/L, normal < 40) upon neonatal screening who was treated as having CAH up to the age of 8 months. He was brought to us for molecular diagnosis. Medication was gradually suspended and serum 17-OHP dosages mantained normal. The p.V281L mutation was found in compound heterozygous status with a group of nucleotide alterations located at the 3' end intron 4 and 5' end exon 5 corresponding to the splice site acceptor region. Molecular studies continued in order to exclude the possibility of a nonclassical 21-OHD form. The group of three nucleotide changes was demonstrated to be a normal variant since they failed to interfere with the normal splicing process upon minigene studies.

A deficiência de 21-hidroxilase (21-OHD) é uma doença autossômica recessiva que contribui com mais de 90 por cento dos casos de hiperplasia congênita da adrenal. O teste de dosagem de 17-hidroxiprogesterona (17-OHP) por radioimunoensaio em amostras de sangue colhidas em papel de filtro tem sido o método mais usado nos programas de triagem neonatal. No entanto, essa triagem pode apresentar alto número de falso-positivos pelo fato de os recém-nascidos prematuros apresentarem dosagens mais elevadas deste esteróide. Apresentamos aqui os estudos moleculares de uma criança, sexo masculino, nascida pré-termo (IG = 30 sem; peso = 1.390 g) que apresentava valores elevados de 17-OHP sérica (91,2 nmol/L, normal < 40) na triagem neonatal e que foi tratada como portadora da forma clássica da 21-OHD até a idade de 8 meses quando nos foi encaminhada para diagnóstico molecular. A terapia foi, então, gradativamente descontinuada, sendo que as concentrações séricas de 17-OHP se mantiveram normais. A mutação p.V281L foi encontrada em heterozigose composta com um grupo de alterações no terminal 3' do íntron 4 e no terminal 5' do éxon 5 correspondendo à região do sítio aceptor de splicing. A análise do gene CYP21A2 prosseguiu para se excluir a possibilidade de a criança ser afetada com a forma não-clássica de 21-OHD. Pela análise de minigene ficou demonstrado que o grupo de três trocas nucleotídicas não afeta o processo normal de transcrição. Concluindo, a criança é apenas heterozigota da mutação p.V281L sem necessidade de tratamento.