RESUMO
Patients with posttraumatic stress disorder (PTSD) appear to manifest two opposing tendencies in their attentional biases and symptoms. However, whether common neural mechanisms account for their opposing attentional biases and symptoms remains unknown. We here propose a model in which reciprocal inhibition between the amygdala and ventromedial prefrontal cortex (vmPFC) predicts synchronized alternations between emotional under- and overmodulatory states at the neural, behavioral, and symptom levels within the same patients. This reciprocal inhibition model predicts that when the amygdala is dominant, patients enter an emotional undermodulatory state where they show attentional bias toward threat and manifest re-experiencing symptoms. In contrast, when the vmPFC is dominant, patients are predicted to enter an emotional overmodulatory state where they show attentional bias away from threat and avoidance symptoms. To test the model, we performed a behavioral meta-analysis (total N = 491), analyses of own behavioral study (N = 20), and a neuroimaging meta-analysis (total N = 316). Supporting the model, we found the distributions of behavioral attentional measurements to be bimodal, suggesting alternations between the states within patients. Moreover, attentional bias toward threat was related to re-experiencing symptoms, whereas attentional bias away from threat was related with avoidance symptoms. We also found that the increase and decrease of activity in the left amygdala activity was related with re-experiencing and avoidance symptoms, respectively. Our model may help elucidate the neural mechanisms differentiating nondissociative and dissociative subtypes of PTSD, which usually show differential emotional modulatory levels. It may thus provide a new venue for therapies targeting each subtype.
Assuntos
Transtornos de Estresse Pós-Traumáticos , Tonsila do Cerebelo , Emoções , Humanos , Imageamento por Ressonância Magnética , Neuroimagem , Córtex Pré-FrontalRESUMO
BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
Assuntos
Antígenos Glicosídicos Associados a Tumores/genética , Neoplasias da Vesícula Biliar/genética , Indígenas Sul-Americanos/genética , Animais , Antineoplásicos/farmacologia , Líquido Ascítico/metabolismo , Carcinogênese/genética , Testes de Carcinogenicidade , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Chile , Cisplatino/farmacologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Impressões Digitais de DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Células Epiteliais/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Perfilação da Expressão Gênica , Genes erbB-2/genética , Humanos , Queratina-19/genética , Queratina-7/genética , Masculino , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Análise de Sequência de RNA , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , GencitabinaRESUMO
To investigate whether specific obesity/metabolism-related gene expression patterns affect the survival of patients with ovarian cancer. Clinical and genomic data of 590 samples from the high-grade ovarian serous carcinoma (HGOSC) study of The Cancer Genome Atlas (TCGA) and 91 samples from the Australian Ovarian Cancer Study were downloaded from the International Cancer Genome Consortium (ICGC) portal. Clustering of mRNA microarray and reverse-phase protein array (RPPA) data was performed with 83 consensus driver genes and 144 obesity and lipid metabolism-related genes. Association between different clusters and survival was analyzed with the Kaplan-Meier method and a Cox regression. Mutually exclusive, co-occurrence and network analyses were also carried out. Using RNA and RPPA data, it was possible to identify two subsets of HGOSCs with similar clinical characteristics and cancer driver mutation profiles (e.g. TP53), but with different outcome. These differences depend more on up-regulation of specific obesity and lipid metabolism-related genes than on the number of gene mutations or copy number alterations. It was also found that CD36 and TGF-ß are highly up-regulated at the protein levels in the cluster with the poorer outcome. In contrast, BSCL2 is highly up-regulated in the cluster with better progression-free and overall survival. Different obesity/metabolism-related gene expression patterns constitute a risk factor for prognosis independent of the therapy results in the Cox regression. Prognoses were conditioned by the differential expression of obesity and lipid metabolism-related genes in HGOSCs with similar cancer driver mutation profiles, independent of the initial therapeutic response.
Assuntos
Cistadenocarcinoma Seroso/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Metabolismo dos Lipídeos/genética , Obesidade/genética , Neoplasias Ovarianas/genética , Idoso , Análise por Conglomerados , Cistadenocarcinoma Seroso/patologia , Feminino , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Neoplasias Ovarianas/patologia , PrognósticoRESUMO
BACKGROUND: High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histologic type of ovarian cancer. To date, there are no reliable biomarkers to effectively predict patient prognosis. Studies have demonstrated inflammation and tumor infiltrating lymphocytes (TILs) correlate with a bad and good prognosis, respectively. Here, we sought to evaluate systemic inflammation and TILs as early prognostic markers of survival. METHODS: Neutrophil-to-lymphocyte ratio (NLR) and serum Lactate Dehydrogenase (LDH) were used as indicators of systemic inflammation. NLR, serum LDH, tumor infiltrating lymphocytes (TILs), PDL1 and quality of debulking surgery were evaluated as determinants of progression-free survival (PFS) and overall survival (OS) in a cohort of 128 HGSOC patients. RESULTS: Initial univariate analysis showed that systemic inflammation measures (NLR and serum LDH), debulking surgery, and intra-epithelial TILs have a significant impact on both PFS and OS. After adjustment for several variables, multivariate analyses confirmed intraepithelial CD4+ T-cells, systemic inflammation measures, PDL1 and debulking surgery as determinants of better OS and PFS. CONCLUSIONS: Systemic inflammation and TILs are early determinants of OS in HGSOC. Other variables such as the quality of debulking surgery and PDL1 also improve survival of patients. Regarding TIL sub-populations, intraepithelial CD4+ cells are associated to an increase in both PFS and OS. We also confirmed previous reports that demonstrate intraepithelial CD8+ cells correlate with an increase on PFS in ovarian cancer. A combined score using systemic inflammation and TILs may be of prognostic value for HGSOC patients.
Assuntos
Biomarcadores Tumorais/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Ovarianas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Biópsia , Cistadenocarcinoma Seroso , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Inflamação/sangue , Inflamação/mortalidade , Inflamação/patologia , L-Lactato Desidrogenase/sangue , Contagem de Linfócitos , Pessoa de Meia-Idade , Gradação de Tumores , Neutrófilos/imunologia , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Ovário/patologia , Ovário/cirurgia , Prognóstico , Estudos Prospectivos , Estudos RetrospectivosRESUMO
BACKGROUND: An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. METHODS: With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. RESULTS: The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. CONCLUSIONS: We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.
Assuntos
Plaquetas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Tromboplastina/metabolismo , Biomarcadores , Comunicação Celular , Movimento Celular , Fatores Quimiotáticos/metabolismo , Feminino , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/cirurgia , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Tromboplastina/genética , Células Tumorais CultivadasRESUMO
Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.
Assuntos
Inibidores da Angiogênese/farmacologia , Fator Xa/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Fator X/farmacologia , Fator X/uso terapêutico , Fator Xa/uso terapêutico , Proteínas de Helminto/farmacologia , Proteínas de Helminto/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Receptor PAR-1/metabolismo , Peixe-ZebraRESUMO
Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/biossíntese , Dedos de Zinco , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia , Serpinas/biossíntese , Tetraspaninas/genética , Tetraspaninas/metabolismo , Fatores de Transcrição/genética , TranscriptomaRESUMO
Our laboratory has implemented an in vitro assay to estimate the response to chemotherapy in ovarian cancer cells pertaining to individual patients. In two selected patients, we determined the correlation between an in vitro assay of cells from suspected ovarian cancer ascites, with the clinical chemotherapy response. Cancer cells isolated from peritoneal fluid with suspected ovarian cancer were tested for cytotoxicity with corresponding chemotherapy regimens. Circulating Cal25 levels and attending physician consultation determined clinical course and response to chemotherapy. The in vitro assay result correlated with Cal25 levels, progression free survival and attending physician evaluation. The assay predicted correctly the failure of two successive chemotherapy regimes in the first patient, while predicting a favorable clinical response in the second subject.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais/análise , Antígeno Ca-125/análise , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Ovarianas/sangue , Medicina de Precisão , Indução de Remissão , Células Tumorais CultivadasRESUMO
Although obesity-associated metabolic disorders have a negative impact on various cancers, such evidence remains controversial for ovarian cancer. Here, we aimed to evaluate the impact of body composition (BC) and metabolism disorders on outcomes in high-grade serous ovarian cancer (HGSOC). METHODS: We analyzed clinical/genomic data from two cohorts (PUC n = 123/TCGA-OV n = 415). BC was estimated using the measurement of adiposity/muscle mass by a CT scan. A list of 425 genes linked to obesity/lipid metabolism was used to cluster patients using non-negative matrix factorization. Differential expression, gene set enrichment analyses, and Ecotyper were performed. Survival curves and Cox-regression models were also built-up. RESULTS: We identified four BC types and two clusters that, unlike BMI, effectively correlate with survival. High adiposity and sarcopenia were associated with worse outcomes. We also found that recovery of a normal BC and drug interventions to correct metabolism disorders had a positive impact on outcomes. Additionally, we showed that immune-cell-depleted microenvironments predominate in HGSOC, which was more evident among the BC types and the obesity/lipid metabolism cluster with worse prognosis. CONCLUSIONS: We have demonstrated the relevance of BC and metabolism disorders as determinants of outcomes in HGSOC. We have shone a spotlight on the relevance of incorporating corrective measures addressing these disorders to obtain better results.
RESUMO
Chronic inflammation influences the tumor immune microenvironment (TIME) in high-grade serous ovarian cancer (HGSOC). Specifically, cyclooxygenase-2 (COX-2) overexpression promotes cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression. Notably, elevated COX-2 levels in the TIME have been associated with reduced response to anti-CTLA-4 immunotherapy. However, the precise impact of COX-2, encoded by PTGS2, on the immune profile remains unknown. To address this, we performed an integrated bioinformatics analysis using data from the HGSOC cohorts (TCGA-OV, n = 368; Australian cohort AOCS, n = 80; GSE26193, n = 62; and GSE30161, n = 45). Employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms, we concluded that COX-2 was linked to immune cell ecosystems associated with shorter survival, cell dysfunction and lower NK cell effector cytotoxicity capacity. Next, we validated these results by characterizing circulating NK cells from HGSOC patients through flow cytometry and cytotoxic assays while undergoing COX-2 and CTLA-4 blockade. The blockade of COX-2 improved the cytotoxic capacity of NK cells against HGSOC cell lines. Our findings underscore the relevance of COX-2 in shaping the TIME and suggest its potential as a prognostic indicator and therapeutic target. Increased COX-2 expression may hamper the effectivity of immunotherapies that require NK cell effector function. These results provide a foundation for experimental validation and clinical trials investigating combined therapies targeting COX-2 and CTLA-4 in HGSOC.
RESUMO
Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.
Assuntos
Coagulação Sanguínea , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Progesterona/metabolismo , Tromboplastina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator VIIa/metabolismo , Fator X/metabolismo , Feminino , Humanos , Microdomínios da Membrana/metabolismo , Invasividade Neoplásica , Transporte Proteico , Receptores de Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
Increased glucose uptake as a principal energy source is a requirement for the continued survival of tumour cells. Facilitative glucose transporter-1 (GLUT1) and -3 (GLUT3) have been previously shown to be present and regulated in breast cancer cells and are associated with poor patient prognosis. In cancer cells, the cAMP secondary messenger pathway is known to potentiate described glucose transporter activators and regulate cell fate. However, no regulation of the glucose transporters in breast cancer cells by cAMP has previously been examined. Herein, we determined in the well-characterized breast cancer cell line ZR-75, if the cAMP analogue 8-br-cAMP was capable of regulating GLUT1 and GLUT3 expression and thus glucose uptake. We demonstrated that 8-br-cAMP transiently up-regulates GLUT3 mRNA levels. The use of actinomycin-D and the cloning of 1,200 bp upstream of the human GLUT3 promoter demonstrated that this regulation was transcriptional. Immunocytochemistry and Western blotting confirmed that the increase in mRNA was reflected by an increase in protein levels. No notable regulation of GLUT1 in the presence of 8-br-cAMP was detected. Finally, we determined using the non-metabolizable glucose analogue 2-DOG if this up-regulation in GLUT3 increased glucose uptake. We observed the presence of two uptake components, one corresponding to the Km of GLUT1/4 and the other to GLUT3. A doubling in the uptake velocity was observed only at the Km corresponding to GLUT3. In conclusion, we demonstrate and characterize for the first time, an up-regulation of GLUT3 mRNA, protein and glucose uptake by the cAMP pathway in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 3/metabolismo , Glucose/metabolismo , Transdução de Sinais , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Transporte Biológico/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Transportador de Glucose Tipo 3/genética , Humanos , Imuno-Histoquímica , Progesterona/farmacologia , RNA Mensageiro/metabolismoRESUMO
Despite numerous endometrial cancer cell lines, little is know about the progression and transition of primary cultured endometrial tumours. Herein, a stage I grade III endometrial adenocarcinoma was maintained in primary culture and the phenotypic and protein expression changes were observed in relation to passage number. At early passage numbers, cultured human endometrial cancer (CHEC) cells displayed classic epithelial cell morphology, growing in groups in a glandular structure and staining positive for cytokeratin. However, with increasing passage number, CHEC cells changed in morphology to display a stromal phenotype which was accompanied by a significant reduction in cytokeratin and increases in alpha-actin and vimentin expression. Simultaneous culture of stromal cells isolated from the original tumour failed to show the same morphological characteristics or protein expression patterns. We further characterised CHEC cells through a screening of cancer related proteins, among others, caveolin-1 and Tissue factor in comparison with established cancer cell lines and corresponding non-cancerous cells. This report demonstrates that endometrial adenocarcinoma cells in culture can undergo phenotypic and protein expression changes reminiscent of epithelial-mesenchymal transition. This work suggests that primary tumours and cell lines displaying stromal morphologies may have undergone epithelial-mesenchymal transition from an adenocarcinoma origin.
Assuntos
Adenocarcinoma/patologia , Neoplasias do Endométrio/patologia , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , FenótipoRESUMO
Cancers of the reproductive tract account for 12% of all malignancies in women. As previous studies have shown that oestrogen metabolites can cause apoptosis, we characterised the effect of oestrogen and oestrogen metabolites on non-cancerous and cancerous human endometrial cells. Herein, we demonstrate that 2-methoxyoestradiol (2ME), but not 17beta-oestradiol, induces apoptosis in cancer cell lines and primary cultured tumours of endometrial origin. In contrast, 2ME had no effect on cell viability of corresponding normal tissue. This ability of 2ME to induce apoptosis does not require oestrogen receptor activation, but is associated with increased entry into the G2/M phases of the cell cycle and the activation of both the intrinsic and the extrinsic apoptotic pathways. The selective behaviour of 2ME on cancerous as opposed to normal tissue may be due to a reduction in 17beta-hydroxysteroid dehydrogenase type II levels in cancer cells and to a differential down-regulation of superoxide dismutase. Furthermore, we demonstrate that pre-treatment with 2ME enhances the sensitivity of reproductive tract cancer cells to the apoptotic drug tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), without the loss in cell viability to normal cells incurred by currently chemotherapeutic drugs. In conclusion, 2ME, alone or in combination with TRAIL, may be an effective treatment for cancers of uterine origin with minimal toxicity to corresponding healthy female reproductive tissue.
Assuntos
Apoptose , Neoplasias do Endométrio/enzimologia , Estriol/análogos & derivados , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , 17-Hidroxiesteroide Desidrogenases/metabolismo , Antineoplásicos/farmacologia , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Endométrio/efeitos dos fármacos , Endométrio/enzimologia , Estriol/farmacologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.
Assuntos
Modelos Biológicos , Neoplasias Ovarianas/patologia , Feminino , Humanos , Imageamento Tridimensional , Microscopia Confocal , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , Coloração e Rotulagem , Células Tumorais Cultivadas , Microtomografia por Raio-XRESUMO
Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.
Assuntos
Plaquetas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Feminino , Humanos , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Células Tumorais CultivadasRESUMO
Progesterone in hormonal preparations increases the incidence of breast cancer. Tissue factor (TF), the initiator of the extrinsic coagulation pathway, is associated with metastasis in a wide variety of cancers. We demonstrate herein that TF mRNA and protein are up-regulated by progesterone in the breast cancer cell line ZR-75. Epidermal growth factor, also associated with increased breast cancer risk, did not regulate TF. The increase in TF is both rapid and transient; increasing after 6 h, reaching a maximum at 24 h, before decreasing to basal levels at 72 h. Sucrose gradient experiments demonstrated that TF is located in the heavy fraction of the plasma membrane, although caveolin-1 is not expressed in ZR-75. To understand the physiological implications of an increase in TF, we performed coagulation and invasion assays. An increase in TF corresponded to an increase in procoagulant activity. Furthermore, progesterone increased the invasion of ZR-75 cells through a matrigel, an effect that was blocked by an antibody against TF. Because TF expression is associated with an enhanced risk of metastasis, we postulate that the progesterone-dependent up-regulation of TF provides a survival advantage to burgeoning breast cancer cells and may contribute to the increased risk of cancer associated with combined hormone replacement therapy.
Assuntos
Neoplasias da Mama/genética , Progesterona/farmacologia , Tromboplastina/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias do Endométrio , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sacarose , Tromboplastina/isolamento & purificaçãoRESUMO
Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.
Assuntos
Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Regulação Neoplásica da Expressão Gênica , Tromboplastina/metabolismo , Adenocarcinoma/metabolismo , Anticoagulantes/farmacologia , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Coagulantes/metabolismo , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Detergentes/farmacologia , Endométrio/citologia , Endométrio/patologia , Estradiol/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Inibidores da Síntese de Ácido Nucleico/farmacologia , Octoxinol/farmacologia , Progesterona/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Sacarose/farmacologia , Fatores de Tempo , Transfecção , Ultracentrifugação , Regulação para CimaRESUMO
To characterize the molecular mechanism and map the response element used by progesterone (P) to upregulate tissue factor (TF) in breast cancer cells. TF expression and mRNA levels were analyzed in breast cancer ZR-75 and T47D cells, using Western blot and real-time PCR, respectively. Mapping of the TF promoter was performed using luciferase vectors. Progesterone receptor (PR) and specificity protein 1 (Sp1) binding to the TF promoter were analyzed by chromatin immuno precipitation assay. Specific or selective inhibitors were used for the MEK1/2 and the c-Src pathways (UO126 and PP2, respectively). TF mRNA increase peaks at 18 h following P treatment in ZR-75 and T47D cells. P upregulation occurs via a transcriptional mechanism that depends on PR and MEK1/2 activation, PR and Sp1 transcription factors bind to a region in the TF promoter that contains three Sp1 sites. TF mRNA upregulation requires an intact PR proline-rich site (mPRO), but it is independent from c-Src. TF upregulation by P is mediated by Sp1 sites in the TF promoter region. Transcriptional upregulation in breast cancer cells occurs via a new mechanism that requires MEK1/2 activation and the mPRO site but independent of c-Src activity. PR Phosphorylation at serine 294 and 345 is not essential.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Progesterona/fisiologia , Prolina/fisiologia , Receptores de Progesterona/fisiologia , Tromboplastina/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes src/genética , Humanos , Fosforilação , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Regulação para Cima/fisiologiaRESUMO
The evidence linking obesity with ovarian cancer remains controversial. Leptin is expressed at higher levels in obese women and stimulates cell migration in other epithelial cancers. Here, we explored the clinical impact of overweight/obesity on patient prognosis and leptin's effects on the metastatic potential of ovarian cancer cells. We assessed clinical outcomes in 70 ovarian cancer patients (33 healthy weight and 37 overweight) that were validated with an external cohort from The Cancer Genome Atlas (TCGA) database. Progression-free and overall survival rates were significantly decreased in overweight patients. Similarly, a worse overall survival rate was found in TCGA patients expressing higher leptin/OB-Rb levels. We explored serum and ascites leptin levels and OB-Rb expression in our cohort. Serum and ascites leptin levels were higher in overweight patients experiencing worse survival. OB-Rb was more highly expressed in ascites and metastases than in primary tumors. Leptin exposure increased cancer cell migration/invasion through leptin-mediated activation of JAK/STAT3, PI3/AKT and RhoA/ROCK and promoted new lamellipodial, stress-fiber and focal adhesion formation. Leptin also contributed to the maintenance of stemness and the mesenchymal phenotype in ovarian cancer cells. Our findings demonstrate that leptin stimulated ovarian cancer cell migration and invasion, offering a potential explanation for the poor prognosis among obese women.