Luciana Nogueira de Sousa Andrade

MT‐1 melatonin receptor expression increases the antiproliferative effect of melatonin on S‐91 murine melanoma cells

Abstract:  Melatonin, a derivative of tryptophan that is present in all vertebrates, was first described in bovine pineal gland. It is known that melatonin is a highly conserved molecule, present also in unicellular organisms and plants. Several effects of melatonin have been described, including receptor‐ and non‐receptor‐mediated actions. Herein, we studied the effects of melatonin on in vitro and in vivo cell proliferation of Cloudman S‐91 murine melanoma cells. We demonstrated that melatonin treatment significantly inhibits S‐91 melanoma cell proliferation in vitro (EC50 = 10−7 m) as well as reduces tumor growth in vivo. We also demonstrated that melatonin directly increases the activity of the antioxidant enzymes catalase and glutathione peroxidase. These effects are most likely triggered through the direct intracellular action of melatonin, since the presence of receptors could not be demonstrated in this cell line. Expression of MT‐1 melatonin receptor by stable transfection, mediated a dramatic antiproliferative melatonin effect (EC50 = 10−10 m) in S‐91 cells. The expressed receptor is negatively coupled to the adenylyl cyclase/cyclic AMP signaling pathway via Gi protein. These results suggest that expression of the MT‐1 melatonin receptor in melanoma cells is a potential alternative approach to specifically target cells in cancer therapeutic treatment.

Evidence for premature aging due to oxidative stress in iPSCs from Cockayne Syndrome

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here, we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However, iPSCs derived from CSB patients fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover, these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells, regulating the expression of TP53 and TXNIP and ROS production.

Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma

Airway inflammation in asthma involves innate immune responses. Toll‐like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics’ both large and small airways has not been investigated.

Anti-tumor effect of endostatin mediated by retroviral gene transfer in mice bearing renal cell carcinoma

We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the NIH/3T3 fibroblast cell line could inhibit renal tumor growth in vivo. NIH/3T3 cells were transduced with retroviral vectors containing the murine endostatin (ES) gene. SCID mice bearing CaKi‐1 derived tumors were given a subcutaneous injection of either ES‐transduced cells or control cells and were monitored for tumor growth. At the end of the in vivo experiment, the mean tumor volume of treated mice was 51.6 ± 2.4 mm3, while the tumor volume of control was 234.5 ± 14.8 mm3. Microvascular density was significantly decreased on treatment (control 9.79 vs. ES 2.53%, < 0.001) accompanied by a 23‐fold increase in intratur‐moral necrotic area and a 2.94‐fold increase in the apoptotic index, determined by immunohistochemistry with anti‐ activated caspase‐3. Apoptotic cells were found in foci enriched in infiltrating leukocytes. In conclusion, retroviral endostatin gene transfer led to secretion of functional endostatin that was sufficiently active to inhibit tumor angiogenesis and tumor growth. A second mechanism may also be implied in endostatin‐dependent tumor regression, associated with tumor infiltration of leukocytes. Besides its antiangiogenic properties, endostatin may be a promising adjuvant to immunotherapy.—Coutinho, E. L., Nogueira de Sousa Andrade, L., Chammas, R., Morganti, L., Schor, N., Bellini, M. H. Anti‐tumor effect of endostatin mediated by retroviral gene transfer in mice bearing renal cell carcinoma. FASEB J. 21, 3153–3161 (2007)

Biological Applications of a Chimeric Probe for the Assessment of Galectin-3 Ligands:

β1–6 branching of N-linked oligosaccharides has been correlated with the progression of different cancers. The leukoagglutinins of Phaseolus vulgaris (L-PHA) have been used to study this pattern of glycosylation whose biological significance is incompletely understood. The animal lectin, galectin-3, also binds to structures recognized by L-PHA. To develop a functional tool for the in situ identification of this pattern of glycosylation, human galectin-3 was fused to bacterial alkaline phosphatase (gal3/AP). Gal3/AP recognized both A and B blood group saccharides (B>A) and lactosamine derivatives. Gal3/AP recognition depended at least in part on the N-linked oligosaccharides of different glycoproteins. The presence and distribution of galectin-3 ligands were analyzed in both murine and human normal and tumor samples. Loss of apical expression of galectin-3 ligands was commonly found in carcinomas. Endothelial and inflammatory cells were enriched in galectin-3 ligands as compared with tumor cells; thus, gal3/AP is a suitable tool for studying tumor micro-environments. Comparative analysis of both gal3/AP and L-PHA binding patterns indicated that although similar, these patterns are not identical. The probe developed was useful for several immunoenzymatic assays and will allow the physiological and clinical significance of the expression pattern of galectin-3 ligands to be established. This manuscript contains online supplemental material at http:/www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 55: 1015–1026, 2007)

Galectin-3 Determines Tumor Cell Adaptive Strategies in Stressed Tumor Microenvironments

Galectin-3 is a member of the β-galactoside-binding lectin family, whose expression is often dysregulated in cancers. While galectin-3 is usually an intracellular protein found in the nucleus and in the cytoplasm, under certain conditions, galectin-3 can be secreted by an yet unknown mechanism. Under stressing conditions (e.g., hypoxia and nutrient deprivation) galectin-3 is upregulated, through the activity of transcription factors, such as HIF-1α and NF-κB. Here, we review evidence that indicates a positive role for galectin-3 in MAPK family signal transduction, leading to cell proliferation and cell survival. Galectin-3 serves as a scaffold protein, which favors the spatial organization of signaling proteins as K-RAS. Upon secretion, extracellular galectin-3 interacts with a variety of cell surface glycoproteins, such as growth factor receptors, integrins, cadherins, and members of the Notch family, among other glycoproteins, besides different extracellular matrix molecules. Through its ability to oligomerize, galectin-3 forms lectin lattices that act as scaffolds that sustain the spatial organization of signaling receptors on the cell surface, dictating its maintenance on the plasma membrane or their endocytosis. Galectin-3 induces tumor cell, endothelial cell, and leukocyte migration, favoring either the exit of tumor cells from a stressed microenvironment or the entry of endothelial cells and leukocytes, such as monocytes/macrophages into the tumor organoid. Therefore, galectin-3 plays homeostatic roles in tumors, as (i) it favors tumor cell adaptation for survival in stressed conditions; (ii) upon secretion, galectin-3 induces tumor cell detachment and migration; and (iii) it attracts monocyte/macrophage and endothelial cells to the tumor mass, inducing both directly and indirectly the process of angiogenesis. The two latter activities are potentially targetable, and specific interventions may be designed to counteract the protumoral role of extracellular galectin-3.

Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200.

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

Protective effect of a Phyllanthus orbicularis aqueous extract against UVB light in human cells

Context: One approach to protect human skin against the dangerous effects of solar ultraviolet (UV) irradiation is the use of natural products, such as photoprotectors. Phyllanthus orbicularis Kunth (Euphorbiaceae) is a Cuban endemic plant used in popular medicine. Its antigenotoxicity effect against some harmful agents has been investigated. However, the effect in ultraviolet B (UVB)-irradiated human cells has not been previously assessed. Objective: The protective effect of a P. orbicularis extract against UVB light-induced damage in human cells was evaluated. Materials and methods: DNA repair proficient (MRC5-SV) and deficient (XP4PA, complementation group XPC) cell-lines were used. Damaging effects of UVB light were evaluated by clonogenic assay and apoptosis induction by flow cytometry techniques. The extent of DNA repair itself was determined by the removal of cyclobutane pyrimidine dimers (CPDs). The CPDs were detected and quantified by slot-blot assay. Results: Treatment of UVB-irradiated MRC5-SV cells with P. orbicularis extract increased the percentage of colony-forming cells from 36.03 ± 3.59 and 4.42 ± 1.45 to 53.14 ± 8.8 and 14.52 ± 1.97, for 400 and 600 J/m2, respectively. A decrease in apoptotic cell population was observed in cells maintained within the extract. The P. orbicularis extract enhanced the removal of CPD from genomic DNA. The CPDs remaining were found to be about 27.7 and 1.1%, while with plant extract, treatment these values decreased to 16.1 and 0.2%, for 3 and 24 h, respectively. Discussion and conclusion: P. orbicularis aqueous extract protects human cells against UVB damage. This protective effect is through the modulation of DNA repair effectiveness.

ATR suppresses apoptosis after UVB irradiation by controlling both translesion synthesis and alternative tolerance pathways.

ABSTRACT Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase &eegr; (Pol &eegr;) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol &eegr;, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.

Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain.

Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 μg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.