RESUMO
We have determined the interfacial properties of tetrahydrofuran (THF) from direct simulation of the vapor-liquid interface. The molecules are modeled using six different molecular models, three of them based on the united-atom approach and the other three based on a coarse-grained (CG) approach. In the first case, THF is modeled using the transferable parameters potential functions approach proposed by Chandrasekhar and Jorgensen [J. Chem. Phys. 77, 5073 (1982)] and a new parametrization of the TraPPE force fields for cyclic alkanes and ethers [S. J. Keasler et al., J. Phys. Chem. B 115, 11234 (2012)]. In both cases, dispersive and coulombic intermolecular interactions are explicitly taken into account. In the second case, THF is modeled as a single sphere, a diatomic molecule, and a ring formed from three Mie monomers according to the SAFT-γ Mie top-down approach [V. Papaioannou et al., J. Chem. Phys. 140, 054107 (2014)]. Simulations were performed in the molecular dynamics canonical ensemble and the vapor-liquid surface tension is evaluated from the normal and tangential components of the pressure tensor along the simulation box. In addition to the surface tension, we have also obtained density profiles, coexistence densities, critical temperature, density, and pressure, and interfacial thickness as functions of temperature, paying special attention to the comparison between the estimations obtained from different models and literature experimental data. The simulation results obtained from the three CG models as described by the SAFT-γ Mie approach are able to predict accurately the vapor-liquid phase envelope of THF, in excellent agreement with estimations obtained from TraPPE model and experimental data in the whole range of coexistence. However, Chandrasekhar and Jorgensen model presents significant deviations from experimental results. We also compare the predictions for surface tension as obtained from simulation results for all the models with experimental data. The three CG models predict reasonably well (but only qualitatively) the surface tension of THF, as a function of temperature, from the triple point to the critical temperature. On the other hand, only the TraPPE united-atoms models are able to predict accurately the experimental surface tension of the system in the whole temperature range.
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Primary cutaneous mantle cell lymphoma (MCL) is a rare cutaneous proliferation of naive pregerminal CD-5 positive B cells in the skin with no extracutaneous involvement. Overexpression of cyclin D1 is pathognomonic of this condition, and surgery and radiation therapy are the most common therapeutic options. In this case, we describe the clinical, histopathological, immunohistochemical, and molecular characteristics of a new case of primary cutaneous MCL.
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Triple negative breast cancer (TNBC) patients are not likely to benefit from anti-estrogen or anti-HER2 therapy and this phenotype is associated with a more aggressive clinical course and worse clinical outcome. Taking into account the limited treatment possibilities in TNBC, the aim of the present work was to study a potential therapy based on Cetuximab-mediated immune activity by natural killer (NK) cells. We performed in vitro studies on human breast cancer (BC) cell lines, IIB-BR-G, and the in vivo metastatic variant IIB-BR-G MT. The immunohistochemical analysis showed a TNBC phenotype with high but different levels of EGFR expression on each cell line, measured by flow cytometry. DNA sequencing showed that both cell lines have a mutated K-RAS status, 38 G > A at codon 13. Consequently, Cetuximab did not inhibit cellular proliferation or induce apoptosis. We investigated if Cetuximab could trigger immune mechanisms, and we determined that both cell lines treated with 1 µg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity (ADCC), mediated by peripheral blood mononuclear cells (PBMC). At 50:1 effector:target ratio, lytic activity was 34 ± 2% against IIB-BR-G and 27 ± 6% against IIB-BR-G MT cells. PBMC pretreatment with IL-2 allowed reaching 65 ± 3% of Cetuximab-mediated ADCC against IIB-BR-G and 63 ± 6.5% against IIB-BR-G MT. Furthermore, IL-15 pretreatment increased the ADCC up to 71 ± 3% in IIB-BR-G and 79 ± 3.5% in IIB-BR-G MT. We suggest that NK cells are the effectors present in PBMC since they were able to induce ADCC at lower effector:target ratios. Besides, IL-2- and mainly IL-15-induced upregulation of NK activating receptors CD16 and NKG2D and enhanced IFN-γ production. EGFR-expressing TNBC could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could advantageously replace IL-2 in most of its immunologic activities, stimulating the ability to produce IFN-γ, and paralleling the up-regulation of activating receptors.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Sequência de Bases , Neoplasias da Mama , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab , Técnicas de Cocultura , Análise Mutacional de DNA , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-15/fisiologia , Interleucina-2/fisiologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de Progesterona/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas ras/genéticaRESUMO
The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.
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Hormônios/fisiologia , Melanoma/metabolismo , Melanoma/secundário , Receptores Androgênicos/metabolismo , Adulto , Ligação Competitiva , Biópsia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Interpretação Estatística de Dados , Di-Hidrotestosterona/farmacologia , Feminino , Hormônios/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Melanoma/patologia , Melanoma Amelanótico/metabolismo , Melanoma Amelanótico/patologia , Melanoma Amelanótico/secundário , Receptores de Esteroides/metabolismo , Células Tumorais CultivadasRESUMO
A monoclonal antibody (MAb) designated FC-5.01 (IgG2a) was generated that binds to several human carcinomas and malignant melanoma. It has revealed no or very low reactivity with most human normal tissues, except for the fact that FC-5.01 binds to some cells from the neuroendocrine system, macrophages, and some renal proximal convolute tubules with an intracellular pattern. Biochemical studies indicate that FC-5.01 recognizes a heterogeneous glycoprotein (broadband between 30-60 kDa) in melanoma tumors. The epitopes reside in the protein core and are presumably conformational, with disulphide bonds implicated in MAb recognition. The current study presents evidence that MAb FC-5.01 reacts with CD63 antigen (Ag), which has been initially described as a melanoma associated Ag, and is a member of the tetraspan family. Reactivity of MAb FC-5.01 with CD63 was demonstrated by Western blot, immunodepletion assay, and FACS analysis of the CD63-negative melanoma cells (KM3) after transfection with the genomic copy of CD63. The epitope recognized by MAb FC-5.01 was shown to be different from the epitope recognized by another anti-CD63 MAb, ME491, by an inhibition radioimmunoassay. Opposite to what has been stated for MAb ME491, no significant differences were found in CD63 expression between primary and metastatic melanoma using MAb FC-5.01.
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Anticorpos Antineoplásicos/imunologia , Antígenos CD/isolamento & purificação , Antígenos de Neoplasias/isolamento & purificação , Melanoma/imunologia , Glicoproteínas da Membrana de Plaquetas/isolamento & purificação , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Carcinoma/imunologia , Citometria de Fluxo , Humanos , Melanoma/secundário , Radioimunoensaio , Tetraspanina 30 , Distribuição TecidualRESUMO
IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplification was found for c-erbB-2 in the cell line or in IIB-BR-G(NUDE), although this oncogene was amplified in the patient's primary tumor DNA. The differences observed between the patient's tumor, the cell line and the IIB-BR-G(NUDE) tumors are probably due to clonal expansion of cell variants not present in the original tumor. Electron microscopy of IIB-BR-G growing cells revealed epithelial characteristics with abundant dense granules, presumably secretory, distributed all over the cytoplasm and great nuclear pleomorphism. In vitro, IIB-BR-G cells showed a significant number of invading cells by Matrigel assay. After nearly 40 sequential subcutaneous passages of the original xenograft through nude mice, 80% of recipients developed spontaneous metastases, primarily to the lung and lymph nodes. Since this experimental model allowed to analyze changes produced in cancer cells from the primary tumor during adaptation to in vitro and in vivo growth, our results provide novel insights on the behaviour of hormone independent metastatic breast cancer.
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Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Antígenos de Neoplasias/biossíntese , Neoplasias da Mama , Carcinoma Ductal de Mama , Feminino , Amplificação de Genes , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Receptores de Estrogênio/genética , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.
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Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/patologia , Melanoma/patologia , Oligonucleotídeos Antissenso/genética , Osteonectina/genética , Animais , Adesão Celular/genética , Divisão Celular/genética , Regulação para Baixo , Humanos , Melanoma/genética , Melanoma Experimental/genética , Camundongos , Transfecção , Células Tumorais CultivadasRESUMO
SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.
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Melanoma/patologia , Osteonectina/biossíntese , Transformação Celular Neoplásica , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , Humanos , Imuno-Histoquímica , Linfotoxina-alfa/farmacologia , Melanoma/química , Melanoma/secundário , Metástase Neoplásica/genética , Osteonectina/genética , RNA Mensageiro/análise , Células Tumorais Cultivadas/químicaRESUMO
Cytokine gene transfer to tumor cells has been demonstrated to induce tumor rejection in different murine models. However, controversial results were presented for different cytokines. In order to study the antitumorigenic activity that has been proposed for IL-6, the poorly immunogenic melanoma B16 and the colon adenocarcinoma CT26-murine cell lines, were transduced with recombinant retrovirus expressing rat IL-6. In vivo studies showed that IL-6-producing-B 16 cells inoculated s.c. in syngeneic mice, exhibited reduced tumorigenicity compared to vector-transduced B 16 cells. The histology of growing IL-6-producing tumors showed a "pseudo-nodular" pattern which correlated with a strong inhibition of the in vitro invasive capacity of these cells. IL-6-producing-B 16 cells did not develop tumors in athymic nude mice suggesting that the antitumor effect is not mediated by a normal host-T- and B-cell response. In contrast, IL-6-producing CT26 cells grew as tumors in syngeneic mice with a faster growth rate than parental and vector-transduced cells, in accordance with an increased in vitro growth kinetics. These results indicate that IL-6 expression by tumor cells demonstrate different effects depending on the tumor cell model.
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Interleucina-6/genética , Células Tumorais Cultivadas/imunologia , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Linfócitos B/imunologia , Divisão Celular , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Expressão Gênica , Engenharia Genética , Cinética , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Transplante de Neoplasias , Ratos , Linfócitos T/imunologia , Transdução Genética , Transplante Isogênico , Células Tumorais Cultivadas/patologiaRESUMO
Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.