RESUMO
Systemic sclerosis (SSc) is an autoimmune disease characterized by significant vascular alterations and multi-organ fibrosis. Microvascular alterations are the first event of SSc and injured endothelial cells (ECs) may transdifferentiate towards myofibroblasts, the cells responsible for fibrosis and collagen deposition. This process is identified as endothelial-to-mesenchymal transition (EndMT), and understanding of its development is pivotal to identify early pathogenetic events and new therapeutic targets for SSc. In this review, we have highlighted the molecular mechanisms of EndMT and summarize the evidence of the role played by EndMT during the development of progressive fibrosis in SSc, also exploring the possible therapeutic role of its inhibition.
Assuntos
Células Endoteliais/patologia , Endotélio/patologia , Transição Epitelial-Mesenquimal/fisiologia , Escleroderma Sistêmico/patologia , Animais , Fibrose/patologia , Humanos , Miofibroblastos/patologiaRESUMO
Dendritic cells (DCs) are able to orchestrate innate and acquired immunity and can activate and sustain a long-lasting anti-tumor immune response in vivo when used as anti-tumor cell therapy. The selection of the antigen and the choice of its formulation are key points in designing anti-cancer DC-based vaccines. Cell released vesicles/exosomes have been shown to transfer antigens, HLAI/peptide complexes and co-stimulatory molecules to recipient cells. In this study we describe the generation of an allogenic microvesicle cell factory in which the expression of a specific tumor antigen was combined to the expression of co-stimulatory and allogeneic molecules. The DG75 lymphoblastoid cell line was selected as microvesicle producer and transfected with ErbB2, as tumor antigen prototype. The shed microvesicles transferred antigenic components to recipient DCs, increasing their immunogenicity. DC pulsing resulted in cross-presentation of ErbB2 both in HLAI and HLAII compartments, and ErbB2-specific CD8+ T cells from cancer patients were activated by DCs pulsed with vesicle-bound ErbB2. The microvesicle cell factory proposed may represent a source of cell free immunogen to be used for DC-based cancer therapy.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/transplante , Imunoterapia Adotiva , Ativação Linfocitária , Receptor ErbB-2/imunologia , Vesículas Transportadoras/transplante , Antígenos de Neoplasias/genética , Neoplasias da Mama/imunologia , Linhagem Celular , Células Dendríticas/imunologia , Feminino , Antígenos HLA/imunologia , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Receptor ErbB-2/genética , Transfecção , Vesículas Transportadoras/imunologiaRESUMO
In the present paper, serum CA125 modifications in patients undergoing their first IVF cycle were compared with those of patients in their second attempt. A significant increase of this marker was detected in each group of patients at day 14 after embryo transfer. However, the level of CA125 monitored in the patients in their second attempt was significantly higher than that determined in patients undergoing their first ovarian stimulation. This condition does not influence either ovarian response or oocyte and embryo quality. Moreover similar IVF outcome was obtained. Therefore we propose that patients undergoing repeated assisted reproductive technology (ART) cycles may suffer from ovarian surface epithelial damage and/or altered cellular growth rate.
Assuntos
Antígeno Ca-125/sangue , Fertilização in vitro , Adulto , Feminino , Humanos , Indução da Ovulação , Gravidez , Resultado do TratamentoRESUMO
The shedding of membrane vesicles from the cell surface is a vital process considered to be involved in cell-cell and cell-matrix interactions and in tumor progression. By immunoelectron microscopic analysis of surface replicas of 8701-BC human breast carcinoma cells, we observed that membrane vesicles shed from plasma membranes contained densely clustered gelatinase B [matrix metalloproteinase 9 (MMP-9)], beta1 integrins, and human lymphocyte antigen class I molecules. By contrast, alpha-folate receptor was uniformly distributed on the smooth cell membrane and shedding areas. Both cell surface clustering of selected molecules and membrane vesicle release were evident only when cells were cultured in the presence of serum. Vesicle shedding occurred preferentially at the edge or along narrow protrusions of the cell. Specific accumulation of proMMP-9 and active forms of MMP-9 in shed vesicles was also demonstrated by gelatin zymography. In addition, Western blotting analysis showed the presence of a large amount of proMMP-9/tissue inhibitor of metalloproteinase 1 complex. The release of selected areas of plasma membranes enriched with MMP-9 and beta1 integrins indicates that membrane vesicle shedding from tumor cells plays an important role in the directional proteolysis of the extracellular matrix during cellular migration. The presence of human lymphocyte antigen class I antigens suggests a mechanism for tumor cells to escape from immune surveillance.
Assuntos
Neoplasias da Mama/ultraestrutura , Membrana Celular/ultraestrutura , Colagenases/análise , Antígenos de Histocompatibilidade Classe I/análise , Integrina beta1/análise , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Membrana Celular/química , Membrana Celular/patologia , Meios de Cultivo Condicionados , Feminino , Humanos , Metaloproteinase 9 da Matriz , Microscopia Eletrônica , Microscopia Imunoeletrônica , Células Tumorais CultivadasRESUMO
Caveolin (cav-1) and the GPI-anchored alpha-folate receptor (alphaFR) are membrane proteins both found associated to caveolar structures. Several studies in tumor cells independently reported cav-1 downregulation and alphaFR overexpression. Here we analysed the expression of the two molecules in normal and tumor ovarian samples derived from fresh specimens and from cultured cell lines. Whereas normal ovary surface epithelial cells displayed only cav-1 expression, ovarian tumor surgical samples and cell lines (COR, IGROV1, OVCAR3 and OVCA432) displayed high alphaFR and low-level or no cav-1 expression, except those cell lines (SKOV3 and SW626) with the lower alphaFR expression. SKOV3, but not two alphaFR-negative non-ovarian cell lines, exhibited down-regulation of cav-1 expression following stable alphaFR cDNA transfection. Conversely, cav-1 transfection in IGROV1 cells led to downregulated alphaFR expression, together with formation of caveolar structures and reduction of growth capability. Moreover, cav-1 expression was induced in IGROV1 cells by transfection with intracellular anti-alphaFR antibodies to downmodulate alphaFR expression. In cav-1 transfected cells, transcriptional activity of the alphaFR-specific promoter P1 was reduced by 70% and an additional specific DNA-protein complex was identified by gel-shift assay, indicating that cav-1 expression influences alphaFR gene transcription. Together these results support the notion that alphaFR and cav-1 protein expression is reciprocally regulated in ovary cancer cells.
Assuntos
Carcinoma/metabolismo , Proteínas de Transporte/fisiologia , Caveolinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/metabolismo , Receptores de Superfície Celular , Células 3T3 , Animais , Carcinoma/genética , Carcinoma/patologia , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Cavéolas/ultraestrutura , Caveolina 1 , Caveolinas/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Receptores de Folato com Âncoras de GPI , Humanos , Substâncias Macromoleculares , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas , Transfecção , Células Tumorais Cultivadas/metabolismo , Neoplasias Vulvares/patologiaRESUMO
To date, no effective therapeutic treatment allows abrogation of the progression of prostate cancer (PCa) to more invasive forms. One of the major targets for the therapy in PCa can be epidermal growth factor receptor (EGFR), which signals via the phosphoinositide 3'-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, among others. Despite multiple reports of overexpression in PCa, the reliance on activated EGFR and its downstream signalling to the PI3K and/or MAPK/extracellular signal-regulated kinase (ERK) pathways has not been fully elucidated. We reported that the EGFR-selective tyrosine kinase inhibitor gefitinib (ZD1839; Iressa) is able to induce growth inhibition, G(1) arrest and apoptosis in PCa cells and that its effectiveness is associated primarily with phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression (and thus Akt activity). In fact PTEN-negative PCa cells are slowly sensitive to gefitinib treatment, because this molecule is unable to downregulate PI3K/Akt activity. PI3K inhibition, by LY294002 or after PTEN transfection, restores EGFR-stimulated Akt signalling and sensitizes the cells to pro-apoptotic action of gefitinib. The MAPK pathway seems to be involved primarily on cell-growth modulation because dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition (both not cell apoptosis) in PTEN-positive PCa cells and reduced EGF-mediated growth in PTEN-negative cells. Thus the effectiveness of gefitinib requires growth factor receptor-stimulated PI3K/Akt and MAPK signalling to be intact and functional. The loss of the PTEN activity leads to uncoupling of this signalling pathway, determining a partial gefitinib resistance. Moreover, gefitinib sensitivity may be maintained in these cells through its inhibitory potential in MAPK/ERK pathway activity, modulating proliferative EGFR-triggered events. Therefore, our data suggest that the inhibition of EGFR signalling can result in a significant growth reduction and in increased apoptosis in EGFR-overexpressing PCa cells with different modalities, which are regulated by PTEN status, and this may have relevance in the clinical setting of PCa.
Assuntos
Antineoplásicos/uso terapêutico , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Fase G1/efeitos dos fármacos , Gefitinibe , Humanos , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Quinazolinas/farmacologiaRESUMO
Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed G(D3) expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G(D3) and caveolin-1, we used immunogold electron microscopy. This revealed localization of G(D3) in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G(D3) and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both G(D3) and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.
Assuntos
Antígenos Glicosídicos Associados a Tumores/análise , Biomarcadores Tumorais/análise , Caveolinas , Membrana Celular/metabolismo , Gangliosídeos/análise , Proteínas de Membrana/análise , Neoplasias Ovarianas/metabolismo , Caveolina 1 , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Microscopia Imunoeletrônica , Células Tumorais CultivadasRESUMO
Cadherins are Ca(+2)-dependent cell surface proteins involved in the specification of the adhesive properties of cells. They are supposed to play a critical role in morphogenesis and pattern formation. In this paper we show that in the sea urchin embryo there are at least two different cadherins of relative molecular masses 140 and 125 kDa. The 140 kDa cadherin is already present in the fertilized egg and is the sea urchin equivalent of E-cadherin. The 125 kDa cadherin, which can be detected using a broad-spectrum anti-cadherin antibody, appears only at later stages of development. In later embryos these two molecules are distributed differently: E-cadherin is present predominantly in the invaginating endoderm of the gastrula while the 125 kDa protein is present on the cell surface of most epithelia. Consistently with the observed differences in expression and in distribution, antibodies directed against these two cadherins differently perturb sea urchin development. For example, when these antibodies are added to early gastrulas only the antibodies against the 125 kDa component can induce a complete disaggregation of the ectoderm, while anti E-cadherin antibodies induce an abnormal development of the endoderm while the embryo maintains its basic integrity. These results are discussed in view of the need for multiple adhesion receptors during pattern formation and embryogenesis.
Assuntos
Caderinas/biossíntese , Gástrula/química , Ouriços-do-Mar/embriologia , Animais , Anticorpos/farmacologia , Caderinas/química , Adesão Celular/efeitos dos fármacos , Endoderma/efeitos dos fármacos , Regulação da Expressão Gênica , Imuno-Histoquímica , Peso MolecularRESUMO
CXCR4 (fusin) is a chemokine receptor which is involved as a coreceptor in gp120 binding to the cell surface. In this study we provide evidence that binding of gp120 triggers CXCR4 recruitment to glycosphingolipid-enriched microdomains. Scanning confocal microscopy showed a nearly complete localization of CXCR4 within GM3-enriched plasma membrane domains of SupT1 cells and coimmunoprecipitation experiments revealed that CXCR4 was immunoprecipitated by IgG anti-GM3 after gp120 pretreatment. These findings reveal that gp120 binding induces a strict association between CXCR4 and ganglioside GM3, supporting the view that GM3 and CXCR4 are components of a functional multimolecular complex critical for HIV-1 entry.
Assuntos
Gangliosídeo G(M3)/metabolismo , Receptores CXCR4/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia em Camada Fina , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Testes de Precipitina , Ligação ProteicaRESUMO
The ability of a cell to modify the extracellular matrix is important in several pathophysiological alterations including tumorigenesis. Cell transformation is accompanied by changes in the surrounding stroma as a result of the action of specific proteases such as the urokinase plasminogen activator (uPA), which has been associated with invasive potential in many tumor types. In this study, we analyzed the release of vesicle-associated uPA by the aggressive prostatic carcinoma cell line PC3 and the implications of this release for the invasive behaviour of prostatic tumor cells. Zymography and Western blot analysis revealed the presence of vesicle-associated uPA in the high-molecular weight form. Vesicles adhered to and degraded both collagen IV and reconstituted basal membrane (Matrigel), and plasminogen enhanced the degradation in a dose-dependent manner. Addition of membrane vesicles shed by PC3 cells to cultures of the poorly invasive prostate cancer cell line LnCaP enhanced the adhesive and invasive capabilities of the latter, suggesting a mechanism involving substrate recognition and degradation. Together, these findings indicate that membrane vesicles can promote tumor invasion and point to the important role of vesicle-associated uPA in the extracellular compartment.
Assuntos
Invasividade Neoplásica/fisiopatologia , Neoplasias da Próstata/patologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Células 3T3 , Animais , Western Blotting , Colágeno/metabolismo , Meios de Cultivo Condicionados , Combinação de Medicamentos , Humanos , Hidrólise , Laminina , Masculino , Camundongos , Microscopia Imunoeletrônica , Neoplasias da Próstata/metabolismo , Proteoglicanas , Células Tumorais CultivadasRESUMO
We have compared the pattern of surface antigen expression, as detected by monoclonal antibodies (mAbs), in plasma membranes vs shed membrane vesicles of two human breast carcinoma cell lines, MCF-7 and 8701-BC. Antigen expression was detected on cells by immunofluorescence (IF) analysis, whilst, due to their small dimensions, the same technique was not applicable to vesicles. For these structures dot-blot analysis and immunoelectron microscopy (IEM) were employed. When applicable, both cell membranes and membrane vesicles were immunoprecipitated and the precipitate (IP) was analyzed by SDS-PAGE. Cells of both lines expressed HLA class I antigens, epithelial cytokeratins, beta 1 integrins, CEA and the glycoprotein detected by mAb 19.9, but only MCF-7 cells expressed Lewis Y, episialin and globo-H antigens and only 8701-BC cells expressed folate receptor. Membrane vesicles of both cell lines appeared to be rich in beta 1, alpha 3 and alpha 5 integrin chains, expressed HLA class I antigens and carried most of the plasma membrane antigens found in the cell membranes. Overall we have analyzed 17 antigens on the two cell lines and on their vesicles. The results obtained for cells (IF and IP) and those for vesicles (dot-blot and IP) were generally concordantly positive or concordantly negative. We obtained a total of 26 clearly concordant combinations on 34 analyses. In three cases we found discordant results, whereas in the remaining combinations we observed slight reactivity and we found difficulties in determining concordance. Discordant results concerned the expression of the following antigens: folate receptors, which were clearly expressed in 8701-BC cells but not detected by dot-blot analysis or IEM on their shed membrane vesicles; neu (c-erb-B2) receptor found in MCF-7 cell membranes but not in their vesicles; and the globo-H antigen recognized by mAb MBr1, detected at low levels on 8701-BC plasma membranes but undetectable on their membrane vesicles. Like vesicles shed in vitro by cultured cells, the vesicles shed in vivo by human breast carcinoma cells could be tagged with several antibodies against tumor-associated antigens. The vesicles shed in vivo were found in association with a fiber network. Some of the fibers had the characteristic fibrin periodicity. These data suggest that tumor markers detected in the circulation of carcinoma patients, at least in part, are carried by shed membrane vesicles. Moreover the observation that membrane vesicles carry both tumor-associated antigens and HLA class I molecules indicate that these structures could in principle present antigens to the immune system.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos de Neoplasias/análise , Antígenos de Superfície/análise , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , Lipossomos/química , Membrana Celular/química , Meios de Cultura , Espaço Extracelular/química , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia Imunoeletrônica , Derrame Pleural Maligno/química , Células Tumorais CultivadasRESUMO
The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and its cell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPA in matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulating the amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differential production of uPA corresponds with the capacity to bind and activate plasminogen. In addition, we provide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize the invasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquire plasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin through exogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observed in several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to the pericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmin generated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the proteolytic cascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chain of uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which may regulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies against uPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growth in uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF) induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic cancer patients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growth of the primary tumor and dissemination of metastatic cells.
Assuntos
Fibrinolisina/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Ativadores de Plasminogênio/metabolismo , Neoplasias da Próstata/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Benzamidinas/farmacologia , Divisão Celular , Colágeno/metabolismo , Combinação de Medicamentos , Ativação Enzimática , Proteínas da Matriz Extracelular/metabolismo , Humanos , Laminina/metabolismo , Masculino , Metaloendopeptidases/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Plasminogênio/administração & dosagem , Plasminogênio/metabolismo , Ativadores de Plasminogênio/antagonistas & inibidores , Neoplasias da Próstata/patologia , Proteoglicanas/metabolismo , Inibidores de Serina Proteinase/farmacologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen alpha-Folate Receptor (alpha-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.
Assuntos
Adenocarcinoma Papilar/enzimologia , Membrana Celular/ultraestrutura , Metaloendopeptidases/metabolismo , Neoplasias Ovarianas/enzimologia , Biomarcadores Tumorais/análise , Western Blotting , Membrana Celular/enzimologia , Feminino , Imunofluorescência , Humanos , Técnicas In Vitro , Metaloendopeptidases/fisiologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Ativadores de Plasminogênio/metabolismo , Células Tumorais CultivadasRESUMO
We recently cloned a cDNA encoding an RNA-binding protein, that we called PIPPin, which is highly enriched in the rat brain and contains two putative double stranded RNA-binding domains (PIP1 and PIP2) and a central cold shock domain (CSD). Here we report that PIPPin is specifically enriched in some pyramidal neurons of the cerebral cortex and in the Purkinje cells of the cerebellum. We also show that PIPPin inhibits translation of H1(o) and H3.3 mRNA in a cell-free system. The results reported suggest that PIPPin down-regulates histone variant expression in the developing rat brain.
Assuntos
Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Proteínas do Tecido Nervoso/metabolismo , Células de Purkinje/metabolismo , Células Piramidais/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas do Tecido Nervoso/análise , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/análise , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
The effect of hyperthermia and bupivacaine, alone and in combination, on the clonogenic activity of a human melanoma cell line was investigated. The time-temperature relationship of exponentially growing cells was defined in the range of 41-45 degrees C. All the survival curves were exponential and the Arrhenius plot was linear over the temperature range tested. The survival curve of bupivacaine-treated cells was also exponential after an initial shoulder. Bupivacaine affected cell survival mainly through an ATP depletion because of deep alterations of mitochondria, essentially due to changes in the physical state of membrane lipids. The analysis of the interaction between hyperthermia and bupivacaine, performed with an isobolar method, demonstrated a synergism of response at all combinations tested, but only with simultaneous exposure. Such a response did not depend on an impairment of the energy-yielding processes, but may be ascribed to combined effects of both agents on cell structure and function. The hyperthermic enhancement achieved by low bupivacaine concentrations allowed to achieve a preestablished cell killing with a reduced exposure time (e.g., 50 min) and with a temperature (42 degrees C) generally accepted as clinically achievable. Therefore, a combined modality in which local treatment with bupivacaine was coupled to local heating could result in high local damage with reduced systemic complications.
Assuntos
Bupivacaína/farmacologia , Hipertermia Induzida , Melanoma/metabolismo , Bupivacaína/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Temperatura Alta , Humanos , Melanoma/patologia , Termodinâmica , Células Tumorais CultivadasRESUMO
We have established an ovarian cancer cell line (CABA I) from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. The epithelial origin of the cell line was confirmed by morphology and by immunofluorescence analysis using anticytokeratin antibodies. Ultrastructural analysis revealed a very irregular membrane surface and a clear cytoplasm rich in electron-lucent vesicles. CABA I cells grow rapidly in culture (doubling time 18 h) in an anchorage-independent manner. Exogenously added beta-estradiol and epidermal growth factor (EGF) treatments did not influence cell growth rate. FACS analysis to determine the phenotypic profile of tumor-associated antigen, membrane receptor, and adhesion molecule expression indicated that the cell line was positive for different members of the c-erbB family, for alpha 6 and beta 1 integrin receptors, and intensively positive for HLA class I antigens and the folate receptor. Molecular characterization revealed no mutations for c-myc and c-k-ras genes, but did detect an exon 5 mutation in the p53 gene. CABA I cells grew poorly as heterotransplants in nude mice, and tumors showed long latency periods. Because early (15-20) and late (55-60) passage cells maintain the same growth and phenotypic characteristics, the CABA I cell line might provide a good in vitro model system to investigate the cellular and molecular events involved in ovarian carcinogenesis.
Assuntos
Carcinoma/patologia , Neoplasias Ovarianas/patologia , Células Tumorais Cultivadas , Animais , Antígenos de Neoplasias/análise , Ascite , Bandeamento Cromossômico , Feminino , Citometria de Fluxo , Humanos , Camundongos , Microscopia Eletrônica , Pessoa de Meia-Idade , Transplante de NeoplasiasRESUMO
The rate of membrane vesicle shedding by tumor cells is probably related to their invasive capability. In order to verify whether the vesicle amount could be utilized as a marker of different pathologies, we analyzed biological fluids obtained from 33 patients with gynecological diseases. In fluids of benign serous cysts, vesicle content was extremely low; in cystoadenomas and fibromas generally it was low. On the contrary, large amounts of vesicles were found in malignant tumor fluids. Gelatin zymographies showed the presence of MMP-2 and MMP-9 in all vesicles except in those recovered from fluids of some serous cysts. A positive correlation between tumor malignancy and both vesicle-amount and vesicle-associated MMP-2 activity was noticed. We also analyzed vesicle content in ascitic fluids recovered from two carcinomas at different times during clinical treatment. In both cases, tumor progression, not monitored by Ca 125 levels, was pointed out by an increased amount of vesicles in ascites. These findings suggest that vesicle content in biological fluids could represent a new useful marker of tumor aggressiveness and tumor progression.
Assuntos
Biomarcadores Tumorais/análise , Líquidos Corporais/metabolismo , Neoplasias Ovarianas/metabolismo , Líquido Ascítico/metabolismo , Antígeno Ca-125/análise , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Densitometria , Progressão da Doença , Feminino , Humanos , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Microscopia Eletrônica , Neoplasias Ovarianas/patologia , Prognóstico , Fatores de TempoRESUMO
In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.
Assuntos
Gangliosídeo G(M2)/química , Gangliosídeo G(M3)/química , Células 3T3 , Animais , Anticorpos Monoclonais , Membrana Celular/química , Membrana Celular/ultraestrutura , Imunofluorescência , Gangliosídeo G(M2)/imunologia , Gangliosídeo G(M3)/imunologia , Humanos , Linfócitos/química , Camundongos , Microscopia Confocal , Microscopia Imunoeletrônica , Polietilenoglicóis , Solubilidade , Células Tumorais CultivadasRESUMO
Paclitaxel (PTX) is a potent anti-neoplastic agent that is highly effective in treating ovarian cancer. Nevertheless, the emergence of PTX resistance has limited the control of this disease. To gain insight into the molecular alterations accompanying drug resistance in ovarian cancer, we generated a new stable PTX-resistant ovarian carcinoma cell line. CABA I cells, which display an intrinsic PTX resistance (IC50 = 800 ng/ml), were subjected to continuous exposure to PTX. From the residual surviving cells, the highly PTX-resistant line CABA-PTX (IC50 = 256000 ng/ml) was generated and stably maintained in vitro. Analysis of beta-tubulin expression indicated that only the HM40 and Hbeta9 isotypes were expressed in both parental and resistant cells. No specific point mutations in the HM40 were detected in either cell line, but expression levels of this isotype were significantly reduced (40%) in CABA-PTX cells. Hbeta9 levels were unchanged. In those cells, PTX resistance was associated with cross-resistance to vinblastine but not to methotrexate or 5-fluorouracil. Verapamil treatment did not reverse the intrinsic drug resistance of parental cells, but partially modulated the sensitivity of CABA-PTX cells to PTX and induced total sensitivity to vinblastine. No changes in the cell surface expression of the drug efflux pumps MRP1, MRP2 and P-glycoprotein were observed. PTX influx, monitored using a fluorescent drug derivative, was significantly reduced and delayed in CABA-PTX cells as compared to the parental cells. Together, these findings suggest that more than one mechanism is involved in PTX resistance, making CABA-PTX cell line a potentially valuable in vitro tool to study multifactorial acquired drug resistance in ovarian cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Fluoruracila/farmacologia , Humanos , Concentração Inibidora 50 , Metotrexato/farmacologia , Proteínas Mitocondriais/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fenótipo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sais de Tetrazólio/farmacologia , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Verapamil/farmacologia , Vimblastina/farmacologiaRESUMO
We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.