RESUMO
Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.
Assuntos
Coriocarcinoma/metabolismo , Regulação da Expressão Gênica , Placenta/metabolismo , Primeiro Trimestre da Gravidez/metabolismo , Esteroide Hidroxilases/metabolismo , Esteroides/metabolismo , Trofoblastos/metabolismo , Adulto , Células Cultivadas , Coriocarcinoma/patologia , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Placenta/citologia , Gravidez , Esteroide Hidroxilases/genética , Trofoblastos/citologiaRESUMO
BACKGROUND: Gestational disorders such as intrauterine growth restriction (IUGR) and pre-eclampsia (PE) are main causes of poor perinatal outcomes worldwide. Both diseases are related with impaired materno-fetal nutrient transfer, but the crucial transport mechanisms underlying IUGR and PE are not fully elucidated. In this study, we aimed to identify membrane transporters highly associated with transplacental nutrient deficiencies in IUGR/PE. RESULTS: In silico analyses on the identification of differentially expressed nutrient transporters were conducted using seven eligible microarray datasets (from Gene Expression Omnibus), encompassing control and IUGR/PE placental samples. Thereby 46 out of 434 genes were identified as potentially interesting targets. They are involved in the fetal provision with amino acids, carbohydrates, lipids, vitamins and microelements. Targets of interest were clustered into a substrate-specific interaction network by using Search Tool for the Retrieval of Interacting Genes. The subsequent wet-lab validation was performed using quantitative RT-PCR on placentas from clinically well-characterized IUGR/PE patients (IUGR, n = 8; PE, n = 5; PE+IUGR, n = 10) and controls (term, n = 13; preterm, n = 7), followed by 2D-hierarchical heatmap generation. Statistical evaluation using Kruskal-Wallis tests was then applied to detect significantly different expression patterns, while scatter plot analysis indicated which transporters were predominantly influenced by IUGR or PE, or equally affected by both diseases. Identified by both methods, three overlapping targets, SLC7A7, SLC38A5 (amino acid transporters), and ABCA1 (cholesterol transporter), were further investigated at the protein level by western blotting. Protein analyses in total placental tissue lysates and membrane fractions isolated from disease and control placentas indicated an altered functional activity of those three nutrient transporters in IUGR/PE. CONCLUSIONS: Combining bioinformatic analysis, molecular biological experiments and mathematical diagramming, this study has demonstrated systematic alterations of nutrient transporter expressions in IUGR/PE. Among 46 initially targeted transporters, three significantly regulated genes were further investigated based on the severity and the disease specificity for IUGR and PE. Confirmed by mRNA and protein expression, the amino acid transporters SLC7A7 and SLC38A5 showed marked differences between controls and IUGR/PE and were regulated by both diseases. In contrast, ABCA1 may play an exclusive role in the development of PE.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Retardo do Crescimento Fetal/patologia , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Placenta/patologia , Pré-Eclâmpsia/patologia , Transportador 1 de Cassete de Ligação de ATP/genética , Adulto , Sistema y+L de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/genética , Estudos de Casos e Controles , Biologia Computacional/métodos , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Recém-Nascido , Placenta/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Gravidez , Adulto JovemRESUMO
BACKGROUND: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. RESULTS: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. CONCLUSIONS: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.
Assuntos
Bases de Dados Genéticas , Modelos Biológicos , Antineoplásicos/farmacologia , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Análise de Componente PrincipalRESUMO
Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.
RESUMO
Membrane transporters and channels (collectively the transportome) govern cellular influx and efflux of ions, nutrients, and drugs. We used oligonucleotide arrays to analyze gene expression of the transportome in 60 human cancer cell lines used by the National Cancer Institute for drug screening. Correlating gene expression with the potencies of 119 standard anticancer drugs identified known drug-transporter interactions and suggested novel ones. Folate, nucleoside, and amino acid transporters positively correlated with chemosensitivity to their respective drug substrates. We validated the positive correlation between SLC29A1 (nucleoside transporter ENT1) expression and potency of nucleoside analogues, azacytidine and inosine-glycodialdehyde. Application of an inhibitor of SLC29A1, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of these two drugs, indicating that SLC29A1 plays a role in cellular uptake. Three ABC efflux transporters (ABCB1, ABCC3, and ABCB5) showed significant negative correlations with multiple drugs, suggesting a mechanism of drug resistance. ABCB1 expression correlated negatively with potencies of 19 known ABCB1 substrates and with Baker's antifol and geldanamycin. Use of RNA interference reduced ABCB1 mRNA levels and concomitantly increased sensitivity to these two drugs, as expected for ABCB1 substrates. Similarly, specific silencing of ABCB5 by small interfering RNA increased sensitivity to several drugs in melanoma cells, implicating ABCB5 as a novel chemoresistance factor. Ion exchangers, ion channels, and subunits of proton and sodium pumps variably correlated with drug potency. This study identifies numerous potential drug-transporter relationships and supports a prominent role for membrane transport in determining chemosensitivity. Measurement of transporter gene expression may prove useful in predicting anticancer drug response.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Canais Iônicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Canais Iônicos/genética , Células K562 , Proteínas de Neoplasias/genética , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNARESUMO
BACKGROUND: The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. RESULTS: We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8), the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff < or = 0.05) of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p < or = 0.05) along the anterior-posterior axis was observed. CONCLUSION: All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the absorption of carrier-mediated compounds in the different intestinal segments. The most pronounced differences can be expected between the adjoining segments ileum and colon, but the differences between the other adjoining segments are not negligible. Finally, for the examined genes, profiles measured in whole intestinal tissue extracts are representative of epithelial cell-only gene expression.
Assuntos
Perfilação da Expressão Gênica/métodos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Transcrição Gênica , Análise de Variância , Animais , Transporte Biológico , Colo/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Íleo/metabolismo , Lasers , Masculino , Camundongos , Microdissecção , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição TecidualRESUMO
Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvß5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvß5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvß5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.
Assuntos
Neoplasias Colorretais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Vitronectina/metabolismo , Quinases da Família src/metabolismo , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HEK293 , Células HT29 , Células Endoteliais da Veia Umbilical Humana , Humanos , Invasividade Neoplásica , Transdução de Sinais , TranscriptomaRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.
Assuntos
Proliferação de Células , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Meduloblastoma/metabolismo , MicroRNAs/genética , Receptores de OSM-LIF/metabolismo , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Meduloblastoma/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de OSM-LIF/genéticaRESUMO
BCL9/9L proteins enhance the transcriptional output of the ß-catenin/TCF transcriptional complex and contribute critically to upholding the high WNT signaling level required for stemness maintenance in the intestinal epithelium. Here we show that a BCL9/9L-dependent gene signature derived from independent mouse colorectal cancer (CRC) models unprecedentedly separates patient subgroups with regard to progression free and overall survival. We found that this effect was by and large attributable to stemness related gene sets. Remarkably, this signature proved associated with recently described poor prognosis CRC subtypes exhibiting high stemness and/or epithelial-to-mesenchymal transition (EMT) traits. Consistent with the notion that high WNT signaling is required for stemness maintenance, ablating Bcl9/9l-ß-catenin in murine oncogenic intestinal organoids provoked their differentiation and completely abrogated their tumorigenicity, while not affecting their proliferation. Therapeutic strategies aimed at targeting WNT responses may be limited by intestinal toxicity. Our findings suggest that attenuating WNT signaling to an extent that affects stemness maintenance without disturbing intestinal renewal might be well tolerated and prove sufficient to reduce CRC recurrence and dramatically improve disease outcome.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Prognóstico , Deleção de Sequência , Fatores de Transcrição , Transcriptoma , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Variação Genética/genética , Mucosa Intestinal/metabolismo , Intestinos/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Colo/química , Colo/metabolismo , Sistemas Computacionais , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Perfilação da Expressão Gênica/veterinária , Imuno-Histoquímica/métodos , Imuno-Histoquímica/veterinária , Mucosa Intestinal/química , Intestino Delgado/química , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , Reação em Cadeia da Polimerase/veterinária , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossínteseRESUMO
The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Armazenamento e Recuperação da Informação/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Mama/genética , Documentação , Feminino , Regulação da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Análise de Sequência de DNA/métodos , SoftwareRESUMO
Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. Sentinel cells, including epithelial cells, lumenal macrophages, and intraepithelial dendritic cells, continuously sense the environment and coordinate defenses for the protection of mucosal tissues. The mucosal epithelial cells are crucial actors in coordinating defenses. They sense the outside world and respond to environmental signals by releasing chemokines and cytokines that recruit inflammatory and immune cells to control potential infectious agents and to attract cells able to trigger immune responses. Among immune cells, dendritic cells (DC) play a key role in controlling adaptive immune responses, due to their capacity to internalize foreign materials and to present antigens to naive T and B lymphocytes, locally or in draining organized lymphoid tissues. Immune cells recruited in epithelial tissues can, in turn, act upon the epithelial cells and change their phenotype in a process referred to as epithelial metaplasia.
Assuntos
Adaptação Fisiológica/imunologia , Células Dendríticas/imunologia , Imunidade nas Mucosas , Animais , Quimiocinas/imunologia , Citocinas/imunologia , Células Epiteliais/imunologia , Humanos , Inflamação/imunologiaRESUMO
Insulin-like growth factor (IGF-I) is a 7648-Da polypeptide consisting of 70 amino acids. Clinically, IGF-I might be used in type II diabetes, which requires a life-long treatment. Therefore, delivery routes other than parenteral injections are highly desirable. For convenience, the peroral route is the most attractive. Therefore, in an attempt to answer the feasibility of oral delivery of IGF-I we examined the metabolism of this polypeptide in the gut in the presence of crude porcine pancreatic enzymes (CPPE) and flushings of the small and large intestine from pig, rat, and dog. Moreover, incubation studies with purified pancreatic enzymes that are present in the intestine were performed to determine the most active enzymes responsible for the intestinal cleavage of IGF-I. IGF-I was mainly degraded by chymotrypsin (t(1/2) = 2.7 min) and trypsin (t(1/2) = 34.6 min), whereas in the presence of aminopeptidase M and carboxypeptidase A IGF-I was stable up to 90 min. IGF-I was degraded in flushings from the jejunum, ileum, and colon. However, there were no significant differences in the stability of IGF-I between the examined intestinal segments. The addition of serine protease inhibitors such as a combination of aprotinin, soybean trypsin inhibitor, and Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as casein profoundly improved the stability. Because we were able to improve the stability of IGF-I in vitro in all species at the same degree we speculate that a similar extension of half-life might also be possible in the human intestinal system.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Animais , Caseínas/metabolismo , Cães , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Fezes/química , Fator de Crescimento Insulin-Like I/farmacocinética , Intestino Grosso/enzimologia , Intestino Delgado/enzimologia , Pâncreas/enzimologia , Ratos , Serina Endopeptidases/metabolismo , SuínosRESUMO
Carrier-mediated transport across membranes plays an important role in drug and nutrient absorption. However, relevant transporters remain largely unknown for most substrates. Their identification requires global analysis of expressed mRNAs in intestinal tissues. Microarray technologies capable of measuring mRNA profiles have proven useful in detecting the expression of genes encoding transporters and ion channels in intestines and Caco-2 cells. This colon carcinoma cell line with characteristics of absorptive enterocytes serves as a common model for drug absorption studies. Gene expression patterns of membrane transporters and channels define the cell's overall transport capacity. Moreover, transporter mRNA profiles provide a basis for assessing drug-drug and drug-food interactions in intestinal absorption. To determine relevant transporters for any given substrate, chemogenomic methods have emerged correlating mRNA expression in multiple tissues to drug transport or response. The resultant drug-transporter databases permit the search for transporter-drug relationships at a genomic scale.
Assuntos
Genômica , Absorção Intestinal/genética , Proteínas de Membrana Transportadoras/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Preparações Farmacêuticas/metabolismo , Animais , Transporte Biológico/genética , Interações Alimento-Droga/genética , Genômica/métodos , Humanos , Proteínas de Membrana Transportadoras/biossínteseRESUMO
During tumor progression cells acquire an altered metabolism, either as a cause or as a consequence of an increased need of energy and nutrients. All four major classes of macromolecules are affected: carbohydrates, proteins, lipids and nucleic acids. As a result of the changed needs, solute carriers (SLCs) which are the major transporters of these molecules are differently expressed. This renders them important targets in the treatment of cancer. Blocking or activating SLCs is one possible therapeutic strategy. For example, some SLCs are upregulated in tumor cells due to the increased demand for energy and nutritional needs. Thus, blocking them and turning off the delivery of fuel or nutrients could be one way to interfere with tumor progression. Specific drug delivery to cancer cells via transporters is another approach. Some SLCs are also interesting as chemosensitizing targets because blocking or activating them may result in an altered response to chemotherapy. In this review we summarize the roles of SLCs in cancer therapy and specifically their potential as direct or indirect targets, as drug carriers or as chemosensitizing targets.
Assuntos
Portadores de Fármacos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Humanos , Modelos BiológicosRESUMO
BACKGROUND: CDX2 plays a key part in the differentiation of Caco-2 cells, a colon carcinoma derived cell line that undergoes spontaneous differentiation. The effect of CDX2 expression in Caco-2 cells over time in culture has not been studied yet on a genome-wide level. METHODS: The impact of CDX2 expression on the genomic profile of Caco-2 cells was studied by transducing cells with CDX2 targeting shRNAs. Knockdown efficiency was assessed on mRNA level and protein level by RTPCR, microarrays, and Western blots. Gene set enrichment analysis was performed to assess regulation of specific gene sets. RESULTS: CDX2 expression had an inhibitory effect on the transcriptional activity of ß-catenin/TCF at early stages of culturing, while at later stages, its role in the trans-activation of target genes specific for small intestinal enterocytes seemed more dominant. CONCLUSIONS: The unique induction of a small intestinal signature upon differentiation in Caco-2 cells seems to be at least partially under the control of CDX2.
Assuntos
Diferenciação Celular , Regulação para Baixo , Enterócitos/citologia , Proteínas de Homeodomínio/metabolismo , Ativação Transcricional , Regulação para Cima , Via de Sinalização Wnt , Fator de Transcrição CDX2 , Células CACO-2 , Quimiotaxia , Bases de Dados Genéticas , Enterócitos/metabolismo , Perfilação da Expressão Gênica , Genômica/métodos , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Cinética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Fatores de Transcrição TCF/antagonistas & inibidores , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: The forkhead box transcription factor FOXQ1 has been shown to be upregulated in colorectal cancer (CRC) and metastatic breast cancer and involved in tumor development, epithelial-mesenchymal transition and chemoresistance. Yet, its transcriptional regulation is still unknown. METHODS: FOXQ1 mRNA and protein expression were analysed in a panel of CRC cell lines, and laser micro-dissected human biopsy samples by qRT-PCR, microarray GeneChip® U133 Plus 2.0 and western blots. FOXQ1 regulation was assayed by chromatin immunoprecipitation and luciferase reporter assays. RESULTS: FOXQ1 was robustly induced in CRC compared to other tumors, but had no predictive value with regards to grade, metastasis and survival in CRC. Prototype-based gene coexpression and gene set enrichment analysis showed a significant association between FOXQ1 and the Wnt pathway in tumors and cancer cell lines from different tissues. In vitro experiments confirmed, on a molecular level, FOXQ1 as a direct Wnt target. Analysis of known Wnt targets identified FOXQ1 as the most suitable marker for canonical Wnt activation across a wide panel of cell lines derived from different tissues. CONCLUSIONS: Our data show that FOXQ1 is one of the most over-expressed genes in CRC and a direct target of the canonical Wnt pathway. It is a potential new marker for detection of early CRC and Wnt activation in tumors of different origins.
Assuntos
Neoplasias Colorretais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Via de Sinalização Wnt/fisiologia , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias Colorretais/genética , Fatores de Transcrição Forkhead/genética , Humanos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Via de Sinalização Wnt/genéticaRESUMO
The cannabinoid G protein-coupled receptors (GPCRs) CB1 and CB2 are expressed in different peripheral cells. Localization of GPCRs in the cell membrane determines signaling via G protein pathways. Here we show that unlike in transfected cells, CB receptors in cell lines and primary human cells are not internalized upon agonist interaction, but move between cytoplasm and cell membranes by ligand-independent trafficking mechanisms. Even though CB receptors are expressed in many cells of peripheral origin they are not always localized in the cell membrane and in most cancer cell lines the ratios between CB1 and CB2 receptor gene and surface expression vary significantly. In contrast, CB receptor cell surface expression in HL60 cells is subject to significant oscillations and CB2 receptors form oligomers and heterodimers with CB1 receptors, showing synchronized surface expression, localization and trafficking. We show that hydrogen peroxide and other nonspecific protein tyrosine phosphatase inhibitors (TPIs) such as phenylarsine oxide trigger both CB2 receptor internalization and externalization, depending on receptor localization. Phorbol ester-mediated internalization of CB receptors can be inhibited via this switch. In primary human immune cells hydrogen peroxide and other TPIs lead to a robust internalization of CB receptors in monocytes and an externalization in T cells. This study describes, for the first time, the dynamic nature of CB receptor trafficking in the context of a biochemical switch, which may have implications for studies on the cell-type specific effects of cannabinoids and our understanding of the regulation of CB receptor cell surface expression.
Assuntos
Receptores de Canabinoides/metabolismo , Animais , Células HL-60 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Células NIH 3T3 , Transporte ProteicoRESUMO
Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.