Deciphering the Functional Status of Breast Cancers through the Analysis of Their Extracellular Vesicles.

Breast cancer (BC) accounts for the highest incidence of tumor-related mortality among women worldwide, justifying the growing search for molecular tools for the early diagnosis and follow-up of BC patients under treatment. Circulating extracellular vesicles (EVs) are membranous nanocompartments produced by all human cells, including tumor cells. Since minimally invasive methods collect EVs, which represent reservoirs of signals for cell communication, these particles have attracted the interest of many researchers aiming to improve BC screening and treatment. Here, we analyzed the cargoes of BC-derived EVs, both proteins and nucleic acids, which yielded a comprehensive list of potential markers divided into four distinct categories, namely, (i) modulation of aggressiveness and growth; (ii) preparation of the pre-metastatic niche; (iii) epithelial-to-mesenchymal transition; and (iv) drug resistance phenotype, further classified according to their specificity and sensitivity as vesicular BC biomarkers. We discuss the therapeutic potential of and barriers to the clinical implementation of EV-based tests, including the heterogeneity of EVs and the available technologies for analyzing their content, to present a consistent, reproducible, and affordable set of markers for further evaluation.

Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival.

Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.

Transcriptome Reprogramming of CD11b+ Bone Marrow Cells by Pancreatic Cancer Extracellular Vesicles.

Pancreatic cancers (PC) are highly metastatic with poor prognosis, mainly due to delayed detection. We previously showed that PC-derived extracellular vesicles (EVs) act on macrophages residing in the liver, eliciting extracellular matrix remodeling in this organ and marked hepatic accumulation of CD11b+ bone marrow (BM) cells, which support PC liver metastasis. We here show that PC-EVs also bind to CD11b+ BM cells and induce the expansion of this cell population. Transcriptomic characterization of these cells shows that PC-EVs upregulate IgG and IgA genes, which have been linked to the presence of monocytes/macrophages in tumor microenvironments. We also report here the transcriptional downregulation of genes linked to monocyte/macrophage activation, trafficking, and expression of inflammatory molecules. Together, these results show for the first time the existence of a PC-BM communication axis mediated by EVs with a potential role in PC tumor microenvironments.

Extracellular Vesicles Shedding Promotes Melanoma Growth in Response to Chemotherapy.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.

Unlike reactivity of mono- and binuclear imine-copper(II) complexes toward melanoma cells via a tyrosinase-dependent mechanism.

The cytotoxicity of a dinuclear imine-copper (II) complex 2, and its analogous mononuclear complex 1, toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2, a tyrosinase mimic, showed much higher activity in comparison to complex 1, and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1, both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2, while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2, in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity.

GD3 ganglioside-enriched extracellular vesicles stimulate melanocyte migration.

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3-ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.

Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death.

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.

Emerging targets for combination therapy in melanomas.

Cutaneous melanomas are often difficult to treat when diagnosed in advanced stages. Melanoma cells adapt to survive in extreme environmental conditions and are among the tumors with larger genomic instability. Here we discuss some intrinsic and extrinsic mechanisms of resistance of melanoma cells to both conventional and target therapies, such as autophagy, adaptation to endoplasmic reticulum stress, metabolic reprogramming, mechanisms of tumor repopulation and the role of extracellular vesicles in this later phenomenon. These biological processes are potentially targetable and thus provide a platform for research and discovery of new drugs for combination therapy to manage melanoma patient treatment.

Mesenchymal stem cells do not prevent antibody responses against human α-L-iduronidase when used to treat mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of α-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. For cell transplantation, 4×10(6) MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. The total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. The antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. The spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of 111In-labeled MSC. In conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases.

Morphological alterations and G0/G1 cell cycle arrest induced by curcumin in human SK-MEL-37 melanoma cells

The aim of this work was to study the effect of curcumin on cell cycle in the human SK-MEL-37 melanoma cell line. In addition, morphological and structural analyses were also performed. Flow cytometric analysis showed a G0/G1 arrest at 5 µM after 24 h exposure and a concentration-dependent increase in the proportion of sub-G0 hypodiploid cells. Typical apoptotic events were also observed by the fluorescence microscopy, transmission and scanning electronic microscopy. Loss of mitochondrial membrane potential was not detected. Results suggested that curcumin could arrest human melanoma cells at G0/G1 phase and induce a mitochondrial-independent apoptotic pathway.

O melanoma é um tipo agressivo de câncer cujo tratamento culmina com o estabelecimento de resistência aos quimioterápicos empregados. Portanto, é importante o desenvolvimento de novos agentes farmacológicos que sejam menos tóxicos e que não provoquem quimiorresistência. As inúmeras propriedades terapêuticas da curcumina vêm sendo confirmadas através de estudos sobre o seu mecanismo de ação em células cultivadas. No presente estudo, empregamos células de melanoma humano da linhagem SK-MEL-37, que desenvolveram resistência in vitro à doxorubicina e cisplatina, drogas normalmente utilizadas na clínica. Investigamos o efeito da curcumina sobre o ciclo celular através de citometria de fluxo. Além disso, análises morfológicas e estruturais também foram realizadas. Os resultados demonstraram que o tratamento com uma concentração de 5 ?M de curcumina provocou uma parada na subfase G0/G1. Além disso, observou-se um aumento dose-dependente na proporção de células hipodiplóides em sub-G0. Eventos apoptóticos típicos foram observados por microscopia de fluorescência, microscopia eletrônica de transmissão e microscopia eletrônica de varredura. Não foi detectada alteração no potencial de membrana mitocondrial. Os resultados indicam que futuros estudos poderão tornar possível a utilização da curcumina como um modulador para agentes quimioterápicos empregados na clínica no tratamento do melanoma.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma.

PURPOSE: We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the anti-hypertensive drug losartan (LOS). RESULTS: We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumor-associated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. CONCLUSIONS: Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Effects of peripheral-type benzodiazepine receptor ligands on Ehrlich tumor cell proliferation.

Peripheral-type benzodiazepine receptors have been found throughout the body, and particularly, in high numbers, in neoplastic tissues such as the ovary, liver, colon, breast, prostate and brain cancer. Peripheral-type benzodiazepine receptor expression has been associated with tumor malignity, and its subcellular localization is important to define its function in tumor cells. We investigated the presence of peripheral-type benzodiazepine receptors in Ehrlich tumor cells, and the in vitro effects of peripheral-type benzodiazepine receptors ligands on tumor cell proliferation. Our results demonstrate the presence of peripheral-type benzodiazepine receptor in the nucleus of Ehrlich tumor cells (85.53+/-12.60%). They also show that diazepam and Ro5-4864 (peripheral-type benzodiazepine receptor agonists) but not clonazepam (a molecule with low affinity for the peripheral-type benzodiazepine receptor) decreased the percentage of tumor cells in G0-G1 phases and increased that of cells in S-G2-M phases. The effects of those agonists were prevented by PK11195 (a peripheral-type benzodiazepine receptor antagonist) that did not produce effects by itself. Altogether, these data suggest that the presence of peripheral-type benzodiazepine receptor within the nucleus of Ehrlich tumor cells is associated with tumor malignity and proliferation capacity.

Papel de dissialogangliosídios na proliferação e morte celular induzida de melanócitos e melanomas in vitro / Role of disialogangliosides in proliferation and induced cell death of melanocytes and melanomas in vitro

Dissialogangliosídios, como GD3 e derivados são marcadores da progressão de melanomas. Para avaliar as possíveis funções desta molécula, transfectamos células de melanócitos com o gene da enzima ST8Sia I, que converte GM3 em GD3. Mostramos que GD3 não interfere na capacidade proliferativa dessas células, porém a expressão de GD3 mostrou-se associada à sobrevivência celular. Melanomas adquirem autonomia quanto às vias dependentes do fator de crescimento de fibroblastos (FGF-1 e -2) / Disialoganglioside GD3 and its derivatives are melanoma progression markers. To evaluate the possible roles of these molecules along melanoma progression, we have transfected the GD3 synthase gene (ST8Sia I) in a melanocyte cell line. Accumulation of GD3 did not confer any proliferative advantage to melanocytes. However, GD3 expression was associated with cell survival. The autonomic growth of melanomas is in part related to a constitutive activation of fibroblast growth factor dependent pathways. GD3 expression did not alter the proliferative response to either FGF-1 or FGF-2...

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

The standart protocol to evaluate the carcionogenic potencial of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative an alternative medium-term bioassay in Wistar rats, also DMBDD assay, based on the paradigm iniation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA) as an official source of evidence of carcinogenity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male wistar rats were separeted into3 groups? the first was treated with no chemicals; the second received five initiaing agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and parafin-fixed and paraffin-embedded tissues after histological analysis) were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This suggests that liver in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS are not among the early molecular alterations occuring in the hepatic carcinogenesis process by the DMBDD protocol in male Wistar rats