RESUMO
The importance of vitamin D in bone and phosphocalcic status is well recognized by the scientific and medical communities; however, recently identified properties of this cholesterol derived molecule, such as immunomodulator and anticancer activities, are yet discussed. Actually, the debate is not so much about the new vitamin D properties, but rather about the optimal concentration required to reach these properties. The difficulty in determining the norms is rendered even more complex by the existence of a vitamin D receptor gene polymorphism. The body pool of this vitamin depends essentially on its endogenous synthesis, but also on its dietary intakes. Many epidemiological studies interested in Vitamin D serum level and cancer suggest a relation between low Vitamin D level and cancer risk, especially in breast and colon adenocarcinomas. In vitro, many studies showed, in different human and animal malignant cell lines, that this molecule exerts anticancer activities: it induces apoptosis and cell differentiation as well as it inhibits proliferation and angiogenesis. This review tries to update the current knowledge on vitamin D and, more particularly, the potential interest of this molecule in cancer prevention and management.
Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/prevenção & controle , Vitamina D/uso terapêutico , Vitaminas/uso terapêutico , HumanosRESUMO
Autologous fat grafting for breast is increasing dramatically. This fat injection needs accurate technical conditions, and shows very good and long-lasting clinical results. Nevertheless, in breast conservative treatment sequellae, fat injection could lead to difficulties in breast imaging, but also there is some concerns about the potential oncologic risks of these procedures.
Assuntos
Tecido Adiposo/transplante , Neoplasias da Mama/cirurgia , Feminino , Humanos , Mastectomia Segmentar , Transplante AutólogoRESUMO
Sleep disordered breathing (SDB) is a prevalent, important nontraditional cardiovascular (CV) risk factor in end-stage renal disease patients. The prevalence of SDB in renal transplant patients is unknown. We compared polysomnographic studies in 163 transplant patients with matched samples in the general population and explored longitudinally the effect of return to dialysis after graft failure on SDB in three consecutive cases. Episodes of nocturnal hypoxemia, average and minimal O(2) saturation overnight in transplant patients did not differ from those in individuals in the general population matched for age, gender and body mass index (BMI). The prevalence of moderate-to-severe SBD in these patients did not exceed the estimated prevalence of the same disturbance in the general population. The respiratory disturbance index in transplant patients was directly associated with BMI (p < 0.001). In the longitudinal study all indicators of SDB coherently increased after transplant failure. The prevalence of SDB in transplant patients does not differ from that in well-matched individuals in the general population. The favorable effect of renal transplantation on CV risk may be at least partially explained by the lack of risk excess for SDB in this population. Longitudinal observations after transplant failure are compatible with the hypothesis that renal transplantation reverses SDB.
Assuntos
Síndromes da Apneia do Sono/terapia , Adulto , Índice de Massa Corporal , Doenças Cardiovasculares/etiologia , Feminino , Rejeição de Enxerto , Humanos , Falência Renal Crônica/complicações , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Oximetria , Polissonografia , Análise de Regressão , Diálise Renal , Fatores de Risco , Síndromes da Apneia do Sono/etiologiaRESUMO
Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.
Assuntos
Adesão Celular , Endotélio Vascular/fisiologia , Matriz Extracelular/fisiologia , Integrinas/fisiologia , Oligopeptídeos/farmacologia , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Animais , Aorta , Bovinos , Adesão Celular/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Fibrinogênio/fisiologia , Fibronectinas/fisiologia , Imunofluorescência , Laminina/fisiologia , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismoRESUMO
One promising avenue towards the development of more selective, better anticancer drugs consists in the targeted delivery of bioactive compounds to the tumor environment by means of binding molecules specific for tumor-associated biomarkers. Eligibility of such markers for therapeutic use implies ideally three criteria : (i) accessibility from the bloodstream, (ii) expression at sufficient level and (iii) no (or much lower) expression in normal tissues. Most current discovery strategies (such as biomarker searching into body fluids) provide no clue as to whether proteins of interest are accessible, in human tissues, to suitable high-affinity ligands, such as systemically delivered monoclonal antibodies. Innovative proteomic technologies are able to identify such accessible biomarkers and represent a key step in the clinical development of such target therapies.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias/terapia , Biomarcadores Tumorais/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias/metabolismo , ProteômicaRESUMO
Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Antígeno 2 Relacionado a Fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sialoglicoproteínas/genética , Fator de Transcrição AP-1/metabolismo , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Sialoproteína de Ligação à Integrina , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/imunologia , RNA Interferente Pequeno/genética , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/imunologiaRESUMO
Zoledronate exhibits antiangiogenic properties in vitro and in vivo. Integrins alphavbeta3 and alphavbeta5 are involved in angiogenesis. Because zoledronate inhibits endothelial cell adhesion, the authors explored the hypothesis that it could alter these integrins recruitment to focal adhesion sites. Human umbilical vein endothelial cells (HUVECs) were treated with zoledronate or with mevalonate pathway intermediates geranylgeraniol (GGOH) and farnesol (FOH). Zoledronate generated a significant decrease in alphavbeta3 and alphavbeta5 expression at HUVEC cell surface using flow cytometry and immunofluorescence. This inhibition was reversed by GGOH but not by FOH. Cells cotreated with zoledronate and GGOH were able to attach to vitronectin through alphavbeta3 and alphavbeta5, as confirmed by the use of specific function-blocking antibodies. The authors showed that zoledronate alters endothelial cell integrin-mediated adhesion. This effect is likely to contribute to the previously demonstrated antiangiogenic effect of zoledronate. Whether this mechanism of action also applies to metastatic tumor cells is under investigation.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Difosfonatos/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imidazóis/farmacologia , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Receptores de Vitronectina/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Diterpenos/farmacologia , Células Endoteliais/citologia , Farneseno Álcool/farmacologia , Adesões Focais/efeitos dos fármacos , Humanos , Ácido Mevalônico/metabolismo , Paxilina/deficiência , Vitronectina/metabolismo , Ácido ZoledrônicoRESUMO
The US FDA approval of broad-spectrum histone deacetylase (HDAC) inhibitors has firmly laid the cancer community to explore HDAC inhibition as a therapeutic approach for cancer treatment. Hitting one HDAC member could yield clinical benefit but this required a complete understanding of the functions of the different HDAC members. Here we explored the consequences of specific HDAC5 inhibition in cancer cells. We demonstrated that HDAC5 inhibition induces an iron-dependent reactive oxygen species (ROS) production, ultimately leading to apoptotic cell death as well as mechanisms of mitochondria quality control (mitophagy and mitobiogenesis). Interestingly, adaptation of HDAC5-depleted cells to oxidative stress passes through reprogramming of metabolic pathways towards glucose and glutamine. Therefore, interference with both glucose and glutamine supply in HDAC5-inhibited cancer cells significantly increases apoptotic cell death and reduces tumour growth in vivo; providing insight into a valuable clinical strategy combining the selective inhibition of HDAC5 with various inhibitors of metabolism as a new therapy to kill cancer cells.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Linhagem Celular Tumoral , Vesículas Citoplasmáticas/metabolismo , Feminino , Glicólise , Xenoenxertos , Humanos , Metabolismo dos Lipídeos , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Musculares/biossíntese , Metástase Neoplásica , Fosforilação OxidativaRESUMO
Histopathological studies of gastrointestinal motility disorders have mainly focused on enteric nerves and interstitial cells of Cajal, but rarely considered the enteric musculature. Here we used both classical and novel smooth muscle markers and transmission electron microscopy (TEM) to investigate muscular alterations in severe colorectal motility disorders. Full-thickness specimens from Hirschsprung's disease, idiopathic megacolon, slow-transit constipation and controls were stained with haematoxylin/eosin (HE) and Masson's trichrome (MT), incubated with antibodies against smooth muscle alpha-actin (alpha-SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin (SM) and histone deacetylase 8 (HDAC8) and processed for TEM. Control specimens exhibited homogeneous immunoreactivity for all antibodies. Diseased specimens showed normal smooth muscle morphology by HE and MT. While anti-alpha-SMA staining was generally normal, immunoreactivity for SMMHC, HDAC8 and/or SM was either absent or focally lacking in Hirschsprung's disease (80%), idiopathic megacolon (75%) and slow-transit constipation (70%). Ultrastructurally, clusters of myocytes with noticeably decreased myofilaments were observed in all diseases. SMMHC and the novel smooth muscle markers SM and HDAC8 often display striking abnormalities linked to the smooth muscle contractile apparatus unnoticed by both routine stainings and alpha-SMA, suggesting specific defects of smooth muscle cells involved in the pathogenesis of gastrointestinal motility disorders.
Assuntos
Doenças do Colo/metabolismo , Doenças do Colo/patologia , Motilidade Gastrointestinal/fisiologia , Músculo Liso/metabolismo , Músculo Liso/patologia , Actinas/metabolismo , Biomarcadores , Proteínas do Citoesqueleto/metabolismo , Histona Desacetilases/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Proteínas Musculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteínas Repressoras/metabolismoRESUMO
The effects of estradiol and two synthetic progestins (ORG2058 and R5020) on the expression of the high-affinity, metastasis-associated laminin receptor in two human breast carcinoma cell lines were examined. The T47D cell line contains estrogen and progesterone receptors, but the MDA-MB 231 cell line lacks both receptors. Treatment of T47D cells with 10(-9) M estradiol alone results in a three-fold increase (P less than or equal to .05) in the steady-state level of laminin receptor mRNA determined by RNA blot analysis as well as in cell-surface, laminin receptor expression that is evaluated by immunofluorescence. No effects of estradiol on the receptor-negative MDA-MB 231 cells were observed. Untreated and steroid-treated MDA-MB 231 cells had higher levels of laminin receptor mRNA than did untreated or estradiol-treated T47D cells. A more dramatic increase (five-fold; P less than or equal to .005) of mRNA and cell-surface expression in T47D cells was observed after treatment with estradiol plus 10(-8) M progestin or with progestin alone. Estradiol treatment also increased chemotaxis and haptotaxis of T47D cells but not of MDA-MB 231 cells to laminin; it had no effect on the attachment of these latter cells to laminin. Interestingly, treatment with estradiol plus progestin or progestin alone significantly increased the attachment of T47D cells to laminin but did not have an effect on either haptotaxis or chemotaxis to laminin. These results suggest that the various cell-laminin interactions are mediated by different mechanisms. The augmentation of laminin receptor mRNA by estrogen and progesterone treatment in hormone receptor-positive cells, but not in cells that lack these receptors, may relate functionally to the difference in the clinical aggressiveness between classes of breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Laminina/metabolismo , Congêneres da Progesterona/farmacologia , Receptores Imunológicos/biossíntese , Movimento Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Laminina/farmacologia , Pregnenodionas/farmacologia , Promegestona/farmacologia , RNA Mensageiro/metabolismo , Receptores Imunológicos/efeitos dos fármacos , Receptores de Laminina , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
BACKGROUND: Stable anchorage of circulating cancer cells to the vasculature is a critical step in the formation of hematogenous metastases. Although the basement membrane glycoprotein laminin clearly plays a crucial role in this event, the exact interactive pathways among cancer cells, laminin, and the vessel wall have not been elucidated. In a previous study, we identified synthetic peptide G, which contains the laminin-binding domain of the 67-kd laminin receptor and which inhibits tumor cell adhesion to endothelial cells. PURPOSE: To assess the role of the interaction between laminin and the 67-kd laminin receptor in hematogenous metastasis formation, we studied the effect of peptide G on melanoma cell behavior in vivo and in vitro. METHODS: The effect of peptide G and control peptides was studied in vivo on lung retention and colonizing potential of murine (B16BL6) and human (A2058) melanoma cells injected intravenously in C57BL/6 and nude mice, respectively. In addition, their effect on cell adhesion and chemotaxis to laminin and on binding of iodine 125-labeled laminin to cells was studied in vitro. RESULTS: In vivo, pretreatment of cells with peptide G resulted in a two- to 10-fold significant increase in the number of experimental lung metastases. A significant relative increase in lung retention of peptide G-treated tumor cells was observed 48 hours after injection, although after 4 hours a partial reduction was observed. In vitro, peptide G significantly increased laminin binding and cancer cell adhesion to laminin and subendothelial matrix, whereas chemotaxis to laminin was significantly inhibited. CONCLUSIONS: Peptide G differentially affected the biological response of cancer cells to laminin. In vitro, it increased laminin binding and cell adhesion to laminin and subendothelial matrix, whereas it inhibited cell chemotaxis to laminin. In vivo, the overall effect of peptide G was an augmentation of lung metastasis. IMPLICATIONS: Our findings suggest that direct adhesion of tumor cells to the subendothelial matrix is a main pathway for hematogenous metastases and that tumor cell-matrix interaction may be more relevant than tumor cell-endothelial cell attachment in this process.
Assuntos
Laminina/metabolismo , Melanoma/patologia , Células Neoplásicas Circulantes , Peptídeos/farmacologia , Precursores de Proteínas , Receptores de Laminina , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Humanos , Melanoma Experimental/patologia , Camundongos , Dados de Sequência MolecularRESUMO
Expression of Gal alpha 1-3Gal cell surface residues has been correlated with the metastatic potential of murine tumor cells. We report that Gal alpha 1-3Gal residues are expressed at the cell surface of malignant human cancer cells, including four cell lines and 50% of the malignant breast specimens obtained by aspiration biopsy. In contrast, all benign breast biopsies and normal cells were Gal alpha 1-3Gal negative. Affinity-purified anti-alpha-galactosyl IgG (anti-Gal) antibody, which specifically recognizes Gal alpha 1-3Gal residues, significantly inhibited cell attachment in two in vitro assays thought to indicate tumor cell extravasation of the circulatory system during the metastatic process: attachment to perfused human umbilical vein endothelium, and attachment to isolated laminin. Since anti-Gal antibody is a natural component of all human sera, we propose that it may be part of the natural antitumor defense system in humans.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Galactosídeos/imunologia , Glicosídeos/imunologia , Células Tumorais Cultivadas/imunologia , Antígenos de Superfície/imunologia , Adesão Celular , Imunofluorescência , Humanos , Metástase Neoplásica , Células Tumorais Cultivadas/citologiaRESUMO
BACKGROUND: Interactions between cells and the basement membrane glycoprotein laminin are altered during colon cancer progression. Colon carcinoma and normal mucosa cells express a variety of laminin-binding proteins, including the 67-kd laminin receptor (67 LR) and a 31-kd human laminin-binding protein (HLBP31) homologous to the 31-kd human IgE-binding protein/galactoside-binding lectin. PURPOSE: To investigate whether various laminin-binding proteins are differentially expressed in human colon carcinoma, we studied messenger RNA (mRNA) levels of the 67 LR and HLBP31 in matched tumor and adjacent normal mucosa samples from a series of 21 patients. METHODS: Total cellular RNA from tumor and normal mucosa was isolated and analyzed by Northern and slot blot hybridization. In addition, HLBP31 protein levels were assessed by the immunoblot technique. Quantitative laminin affinity chromatography was also used to measure the synthesis of HLBP31 protein in five human cancer cell lines. RESULTS: The steady-state mRNA level of HLBP31 was downregulated (i.e., decreased) in 18 of 21 human colon carcinomas compared with the level in their corresponding normal colonic mucosa. On average, the level of HLBP31 mRNA was decreased 50% +/- 30% (+/- SD) in the colon cancers. The mean ratio of colon cancer HLBP31 mRNA to adjacent normal mucosa HLBP31 mRNA was twofold lower in primary tumors of patients with metastases (0.3 +/- 0.2 SD) than in primary tumors of patients free of metastatic lesions (0.6 +/- 0.2 SD). The differences between the two groups of patients were statistically significant (P less than .05, Wilcoxon-Mann-Whitney test). We have previously shown that the ratio of colon cancer 67 LR mRNA to corresponding normal mucosa 67 LR mRNA was increased in the same patient population. When the two ratios (ratio of cancer to normal HLBP31 mRNA and ratio of cancer to normal 67 LR mRNA) were compared, HLBP31 mRNA/67 LR mRNA was significantly lower (P less than .05) in primary tumors with metastases (mean +/- SD, 0.3 +/- 0.2) than in primary cancers without metastases (mean +/- SD, 0.7 +/- 0.5). The steady-state level of HLBP31 mRNA was directly correlated with the amount of HLBP31 protein in both colon tissue samples and human cancer cell lines. CONCLUSION: HLBP31 mRNA expression in colon cancer tissues is modulated inversely to that of 67 LR mRNA expression. The down-regulation of HLBP31 appears to be associated with the metastatic capabilities of colon cancer cells. IMPLICATIONS: Prospective studies on a large cohort should determine if the systematic detection of HLBP31 and 67 LR protein and/or mRNA can be a valuable adjunct in the prognostic evaluation of primary colon cancers.
Assuntos
Carcinoma/química , Neoplasias do Colo/química , Laminina/metabolismo , RNA Mensageiro/análise , Receptores Imunológicos/genética , Sequência de Aminoácidos , Sequência de Bases , Colo/química , DNA/isolamento & purificação , Humanos , Dados de Sequência Molecular , Metástase Neoplásica , Receptores Imunológicos/análise , Receptores de Laminina , Células Tumorais CultivadasRESUMO
The tumor cell laminin receptor is a cell-surface protein that binds laminin with high affinity (Kd = 1.0 nM). The putative ligand-binding domain of the laminin receptor has been molecularly cloned and sequenced. In the present study, we used the predicted amino acid sequence of the laminin receptor to generate synthetic peptide antigens and produced immunoglobulin M (IgM) anti-laminin receptor monoclonal antibodies. The disulfide bond group of the IgM molecule was used to couple the antibodies to the surface of liposomes encapsulating doxorubicin. The anti-laminin receptor monoclonal antibodies coupled to the liposomes bound avidly to the surface of MDA-MB-435S (MDA-435) human breast carcinoma cells, which have high numbers of laminin receptors. These antibody-coupled liposomes exhibited a low degree of binding to Hs 578Bst (Hs 578) normal human breast epithelial cells, which express a low number of laminin receptors. Excess liposomes competed for the binding of unbound laminin to the tumor cell surface, and excess laminin competed for binding with the liposomes. Antibody-coupled liposomes encapsulating doxorubicin were specifically more efficient in inhibiting colony formation by MDA-435 cells in vitro than unbound doxorubicin or liposomes without anti-laminin receptor monoclonal antibodies. Unbound doxorubicin inhibited thymidine uptake by 10%-20% in both Hs 578 and MDA-435 cells, whereas the antibody-coupled liposomes encapsulating doxorubicin inhibited thymidine uptake by 90% in MDA-435 cells but only 15% in Hs 578 cells. Thus, use of anti-laminin receptor monoclonal antibodies coupled with liposomes encapsulating doxorubicin represents a new strategy for selective targeting of doxorubicin to carcinoma cells with exposed laminin receptors.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/administração & dosagem , Laminina/imunologia , Receptores Imunológicos/metabolismo , Neoplasias da Mama/imunologia , Portadores de Fármacos , Humanos , Lipossomos , Receptores Imunológicos/imunologia , Receptores de Laminina , Células Tumorais CultivadasRESUMO
It has been proposed that among the various cell-surface proteins capable of interacting with laminin, the 67-kd high-affinity laminin receptor plays a crucial role during tumor invasion and metastasis. In this study, the expression of laminin-receptor-precursor messenger RNA (mRNA) and 67-kd protein was analyzed in human colon adenocarcinoma. In 22 of 23 patients with colon cancer, we found a 2- to 23-fold increase in levels of laminin-receptor-precursor mRNA in the cancer tissues compared with those in matched normal adjacent colonic mucosa. In 10 of 11 cases studied, the level of 67-kd laminin receptor, detected by affinity-purified anti-laminin-receptor synthetic peptide antibodies on immunoblots of matched tumor and normal tissue extracts, was higher in the colon carcinoma tissue. Immunodetection of laminin receptor in tissue sections using anti-laminin-receptor-peptide antibodies confirmed that the increased expression of laminin receptor was specifically associated with the cancer cells. In a series of 72 paraffin sections of colon lesions, we observed a correlation between the expression of the laminin receptor and the Dukes' classification. Our observations indicate that increased expression of laminin-receptor-precursor mRNA is associated with enhanced levels of the 67-kd laminin receptor as well as with the invasive phenotype of colon carcinoma. Detection of this metastasis-associated gene product may be a valuable adjunct in the evaluation of human colon cancer.
Assuntos
Adenocarcinoma/ultraestrutura , Neoplasias do Colo/ultraestrutura , Receptores Imunológicos/fisiologia , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Neoplasias do Colo/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Receptores de LamininaRESUMO
BACKGROUND: Bone sialoprotein (BSP), a bone matrix protein, was recently found to be expressed ectopically in breast cancer and to have a statistically significant association with poor prognosis and the development of bone metastases in that disease. These data prompted us to investigate whether BSP might also be expressed in human prostate cancer, which often metastasizes to bone, and be predictive for progression risk. METHODS: Tissue sections from 180 patients who had undergone a radical prostatectomy for localized prostate cancer were analyzed immunohistochemically for BSP expression. Biochemical progression was defined as an increasing serum prostate-specific antigen level of 0.5 ng/mL or more. Statistical analysis was used to assess associations between pathologic findings and level of BSP expression, and a Cox proportional hazards model was used to determine which clinical and histologic parameters, including stage, Gleason score, and BSP expression (immunostaining intensity and extent), were independently associated with biochemical progression. All P values were two-sided. RESULTS: Most of the prostate cancer lesions examined (78.9%) expressed detectable levels of BSP, compared with no or low expression in the adjacent normal glandular tissue. A statistically significant association was found between BSP expression and biochemical progression in both univariate and multivariate analyses. After a follow-up interval of 3 years, the biochemical relapse rate was 36.7% (95% confidence interval [CI] = 23.4%-47.7%) in patients whose tumors expressed high levels of BSP compared with 12.1% (95% CI = 2.3%-20.8%) in patients whose tumors expressed no or a low detectable level of the protein (logrank test, P = .0014). BSP expression status could identify those patients at higher risk of biochemical progression (logrank test, P<.05) among patients with moderately differentiated tumors or with pathologically confined tumors. CONCLUSIONS: To our knowledge, this study is the first to demonstrate BSP expression in human prostate cancer and to highlight the protein's statistically significant prognostic value in patients with clinically confined prostate adenocarcinomas.
Assuntos
Adenocarcinoma/química , Osso e Ossos/química , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/química , Sialoglicoproteínas/análise , Idoso , Intervalo Livre de Doença , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Sialoproteína de Ligação à Integrina , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/análise , RNA Neoplásico/análise , Sialoglicoproteínas/genéticaRESUMO
Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 high-affinity receptor (67 LR) could play a major role in this process. Scatchard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000 high-affinity receptors that mediate, at least in part, the attachment of highly invasive melanoma cells. This endothelial cell-melanoma cell interaction was significantly inhibited by soluble laminin and by anti-laminin antibodies. Peptide G, an eicosapeptide derived from the complementary DNA sequence of the 67 LR precursor (IPCNNKGAHSVGLMWWMLAR) that specifically binds to laminin and presumably contains the active ligand-binding site of the receptor, specifically prevented attachment of the melanoma cells to both the bovine aortic endothelial cell monolayer and human umbilical vein endothelium. Thus, peptide G may selectively interfere with the metastatic cascade by inhibiting tumor cell attachment to endothelium via the laminin-67 LR pathway and is a potential new antimetastatic agent.
Assuntos
Endotélio Vascular/fisiopatologia , Laminina/fisiologia , Melanoma/fisiopatologia , Receptores Imunológicos/fisiologia , Sequência de Aminoácidos , Adesão Celular/fisiologia , Humanos , Laminina/genética , Laminina/metabolismo , Dados de Sequência Molecular , Metástase Neoplásica , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Laminina , Células Tumorais CultivadasRESUMO
Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. To date, the molecular mechanism that leads to the deposition of hydroxyapatite in the mammary tissue has not been elucidated. Bone sialoprotein (BSP) is a glycoprotein the expression of which coincides with the appearance of the first hydroxyapatite crystals during bone development. In this study, we report the observation that BSP, a bone matrix protein, is expressed in human mammary cancer cells. Using an immunoperoxidase technique, we studied the expression of BSP in 79 breast lesions, including 28 benign and 51 malignant specimens. Two polyclonal antibodies, one directed against intact human BSP and the other against a synthetic peptide of BSP (residues 277-294), were used and gave identical results. Normal mammary glands expressed undetectable or barely detectable amounts of BSP, and the majority of the benign lesions examined were generally unstained (0) or weakly stained (1+). Most of the breast carcinoma specimens (around 87%) showed a significant increase (P = 0.0001) in BSP expression. Breast carcinomas with microcalcifications had the highest immunoreactivity (2+ or 3+) to BSP antibodies. This is the first demonstration that BSP expression is significantly increased in breast cancer. Expression of BSP by breast cancer cells could play a major role in the deposition of microcalcifications and in the preferred bone homing of breast cancer cells.
Assuntos
Neoplasias da Mama/química , Proteínas de Neoplasias/análise , Sialoglicoproteínas/análise , Mama/química , Carcinoma/química , Carcinoma in Situ/química , Feminino , Fibroadenoma/química , Doença da Mama Fibrocística , Humanos , Sialoproteína de Ligação à IntegrinaRESUMO
Tamoxifen is an antiestrogen used in adjuvant therapy of breast carcinoma and could potentially prevent the development of mammary cancer. While it is widely clinically used, its exact mechanisms of action are not yet fully elucidated. MCF-7/6 cells are estrogen receptor-positive invasive human breast cancer cells with a functionally inactive cell surface E-cadherin. In this study, we report that tamoxifen, and to a lesser extent its metabolites 4-OH-tamoxifen and N-desmethyltamoxifen, restore the function of E-cadherin in MCF-7/6 cells. In an aggregation assay, 10(-6) M tamoxifen significantly increases the aggregation of MCF-7/6 cells. This effect is abrogated by a monoclonal antibody against E-cadherin (HECD-1), is fast (within 30 min), and does not require de novo protein synthesis. Tamoxifen was also found to inhibit the invasion of MCF-7/6 cells in organ culture. Our data is the first demonstration that tamoxifen can activate the function of an invasion suppressor molecule and suggest that the restoration of E-cadherin function may contribute to the therapeutic benefit of tamoxifen in breast cancer patients.