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BACKGROUND: The search for biomarkers to evaluate ovarian cancer (OC) homologous recombination (HR) function and predict the response to therapy is an urgent clinical need to improve the selection of patients who could benefit from platinum- and olaparib (poly-ADP ribose polymerase inhibitors, PARPi)-based therapies. METHODS: We used a large collection of OC patient-derived xenografts (PDXs) (n = 47) and evaluated their HR status based on BRCA1/2 mutations, BRCA1 promoter methylation and the HRDetect score. RAD51 foci were quantified in formalin-fixed, paraffin-embedded untreated tumour specimens by immunofluorescence and the messenger RNA expression of 21 DNA repair genes by real-time PCR. RESULTS: Tumour HR deficiency predicted both platinum and olaparib responses. The basal level of RAD51 foci evaluated in geminin-positive/replicating cells strongly inversely correlated with olaparib response (p = 0.011); in particular, the lower the foci score, the greater the sensitivity to olaparib, while low RAD51 foci score seems to associate with platinum activity. CONCLUSIONS: The basal RAD51 foci score is a candidate predictive biomarker of olaparib response in OC patients as it can be easily translatable in a clinical setting. Moreover, the findings corroborate the importance of OC-PDXs as a reliable tool to identify and validate biomarkers of response to therapy.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Cisplatino/farmacologia , Recombinação Homóloga , Neoplasias Ovarianas/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Platelet thrombospondin-1 (TSP-1) is a major endogenous regulator of growth factor activity in physiological and pathological processes, including tumor onset, progression and angiogenesis. We previously demonstrated that TSP-1 binds to FGF-2, sequestering the growth factor and inhibiting its angiogenic activity. We also identified a non-peptidic antiangiogenic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of TSP-1. AIM: To identify new small molecule inhibitors of FGF2 that recapitulate the structure and functional properties of the FGF-2-binding site of TSP-1, by investigating the chemical space around SM27. MATERIALS AND METHODS: A similarity-based screening of small molecule libraries has been used to identify candidates, followed by docking calculations, and evaluation of the activity of the resulting compounds in biochemical and biophysical assays, to assess interaction with FGF2, and in experimental models of angiogenesis, to assess biological activity. RESULTS: The used integrated approach allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting FGF2 binding to both heparan sulfate proteoglycans and FGFR1. The compounds inhibited FGF2-induced endothelial cell proliferation, vessel sprouting from aortic rings and angiogenesis in the chorioallantoic membrane assay, with improved potency over SM27. CONCLUSIONS: We have identified new compounds that are valuable as FGF inhibitors for potential therapeutic purposes. Moreover, these compounds are useful chemical tools to identify the minimal stereochemical requirements for FGF2 binding and activity to improve the design of new agents for antineoplastic therapy. ACKNOWLEDGEMENT: Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro).
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INTRODUCTION: Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor whose expression is up-regulated by VEGF in microvascular and umbilical vein endothelial cells (EC). Despite this, TFPI-2 has been suggested as anti-angiogenic molecule, due to its ability to inhibit the migration/proliferation of EC induced by VEGF. Nothing is known about the precise mechanism of TFPI-2 function tuning in tumor endothelium. AIM: Aim of this study was to investigate the role of TFPI-2 in tumor vasculature, where angiogenesis and vascular remodeling are fundamental for cancer progression. MATERIALS AND METHODS: Tumor-EC were isolated from ovarian carcinomas and cultured in vitro in presence of factors reproducing the tumor microenvironment (VEGF, FGF-2, EGF). TFPI-2 and PRSS3 silencing was achieved by small interfering RNA (siRNA). Tumor-EC migration was assayed by the wound healing assay. Transcript expression was examined by qRT-PCR. Proteolytic reactions were monitored by western blot. RESULTS: We show that tumor-EC express TFPI-2, the majority of which is released and found anchored in the extracellular matrix. Silencing the expression of TFPI-2 enhances tumor-EC migration, confirming TFPI-2 as an anti-angiogenic molecule. We had previously shown that the cancer vasculature express PRSS3, a trypsin family member able to cleave proteins containing the kunitz-type domains; we reasoned that it could potentially inhibit TFPI-2. Herein, we demonstrate in a cell free system that TFPI-2 directly interacts with and is degraded by active PRSS3. In a more complex biological context, active PRSS3 is able to remove TFPI-2 from the extracellular matrix put down by tumor-EC. Accordingly, silencing PRSS3 causes the extracellular accumulation of TFPI-2 that results in the inhibition of tumor-EC migration. CONCLUSIONS: Our results demonstrate for the first time that TFPI-2 is a direct substrate of PRSS3, which hydrolyses TFPI-2 (most likely at the Kunitz-type domains) blocking its anti migratory capability. The proteolytic inactivation of TFPI-2 by PRSS3 might represent a mechanism favoring cancer by increasing angiogenesis and vascular remodeling. ACKNOWLEDGEMENT: Supported by the Italian Association for Cancer Research (AIRC).
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Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and causes severe body weight loss, with rapid depletion of skeletal muscle. No treatment is available. We analyzed microarray data sets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents but not in those with diabetes, disuse, uremia or fasting. Ingenuity Pathways Analysis indicated that three genes belonging to the C-X-C motif chemokine receptor 4 (CXCR4) pathway were downregulated only in muscles atrophying because of cancer: stromal cell-derived factor 1 (SDF1), adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Notably, we found that, in the Rectus Abdominis muscle of cancer patients, the expression of SDF1 and CXCR4 was inversely correlated with that of two ubiquitin ligases induced in muscle wasting, atrogin-1 and MuRF1, suggesting a possible clinical relevance of this pathway. The expression of all main SDF1 isoforms (α, ß, γ) also declined in Tibialis Anterior muscle from cachectic mice bearing murine colon adenocarcinoma or human renal cancer and drugs with anticachexia properties restored their expression. Overexpressing genes of this pathway (that is, SDF1 or CXCR4) in cachectic muscles increased the fiber area by 20%, protecting them from wasting. Similarly, atrophying myotubes treated with either SDF1α or SDF1ß had greater total protein content, resulting from reduced degradation of overall long-lived proteins. However, inhibiting CXCR4 signaling with the antagonist AMD3100 did not affect protein homeostasis in atrophying myotubes, whereas normal myotubes treated with AMD3100 showed time- and dose-dependent reductions in diameter, until a plateau, and lower total protein content. This further confirms the involvement of a saturable pathway (that is, CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer.
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Caquexia/etiologia , Caquexia/patologia , Quimiocina CXCL12/metabolismo , Atrofia Muscular , Neoplasias/complicações , Neoplasias/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Animais , Benzilaminas , Biomarcadores , Ciclamos , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Humanos , Indóis/farmacologia , Masculino , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Neoplasias/genética , Pirróis/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , SunitinibeRESUMO
We provide an overview of the available information on the distribution of chemotherapeutics in human tumors, highlighting the progress made to assess the heterogeneity of drug concentrations in relation to the complex neoplastic tissue using novel analytical methods, e.g., mass spectrometry imaging. The increase in interstitial fluid pressure due to abnormal vascularization and stiffness of tumor stroma explains the variable and heterogeneous drug concentrations. Therapeutic strategies to enhance tumor drug distribution, thus possibly increasing efficacy, are discussed.
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Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacologia , Diagnóstico por Imagem/métodos , Humanos , Neoplasias/diagnóstico , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
BACKGROUND: Bevacizumab is being incorporated as first-line therapy with standard-of-care chemotherapy on epithelial ovarian carcinoma (EOC). We investigated bevacizumab combined with chemotherapy on tumour progression and mouse survival in EOC xenograft models. METHODS: Bevacizumab was administered concomitantly with cisplatin plus paclitaxel (DDP+PTX), continued after induction (maintenance) or started after chemotherapy. The effect on tumour progression was monitored by bioluminescence imaging (BLI) (1A9-luc xenograft). Tumour dissemination into the peritoneal organs and ascites formation (HOC22 xenograft) was evaluated by histological analysis at the end of treatment (interim) and at euthanasia (survival). The effects on overall survival (OS) were investigated in both EOC models. RESULTS: Bevacizumab with PTX+DDP delayed tumour progression in mice bearing EOC xenografts. OS was significantly extended, with complete responses, by bevacizumab continued after stopping chemotherapy in the HOC22 xenograft. Bevacizumab alone inhibited ascites formation, with only limited effect on tumour burden, but combined with PTX+DDP reduced ascites and metastases. Bevacizumab started after induction with PTX+DDP and maintained was equally effective on tumour progression and survival on 1A9-luc xenograft. CONCLUSION: Bevacizumab combined with chemotherapy not only affected tumour progression, but when administered as maintenance regimen significantly prolonged survival, reducing ascites, and tumour dissemination. We believe our findings are consistent with the clinical results and shed light on the potential effects of this kind of treatment on tumour progression.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Vascular endothelial growth factor (VEGF)-A, placenta growth factor (PlGF) and their corresponding membrane receptors are involved in autocrine and paracrine regulation of melanoma growth and metastasis. Besides the membrane receptors, a soluble form of the VEGF receptor (VEGFR)-1 (sVEGFR-1) has been identified, that behaves both as a decoy receptor, sequestering VEGF-A and PlGF, and as an extracellular matrix (ECM) molecule, promoting endothelial cell adhesion and migration through the interaction with α5ß1 integrin. OBJECTIVES: To analyse whether sVEGFR-1 plays a role during melanoma progression. METHODS: sVEGFR-1 expression was evaluated in a panel of 36 melanoma cell lines and 11 primary human melanocyte cultures by quantitative real-time polymerase chain reaction analysis and in specimens of primary or metastatic melanoma lesions from 23 patients by immunohistochemical analysis. RESULTS: sVEGFR-1 expression was highly upregulated in melanoma cell lines with respect to human melanocytes. Interestingly, cell lines obtained from cutaneous metastases showed a significant reduction of sVEGFR-1 expression, as compared with cell lines derived from primary tumours. These results were confirmed by immunohistochemical analysis of sections from primary skin melanomas and the corresponding cutaneous metastases, suggesting that modulation of sVEGFR-1 expression influences ECM invasion by melanoma cells and metastasis localization. Moreover, we provide evidence that adhesion of melanoma cells to sVEGFR-1 is favoured by the activation of a VEGF-A/VEGFR-2 autocrine loop. CONCLUSIONS: Our data strongly suggest that sVEGFR-1 plays a role in melanoma progression and that low sVEGFR-1/VEGF-A and sVEGFR-1/transmembrane VEGFR-1 ratios might predict a poor outcome in patients with melanoma.
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Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Imuno-Histoquímica , Melanoma/secundário , Reação em Cadeia da Polimerase , Neoplasias Cutâneas/secundárioRESUMO
The clinical success of small-molecule vascular disrupting agents (VDAs) depends on their combination with conventional therapies. Scheduling and sequencing remain key issues in the design of VDA-chemotherapy combination treatments. This study examined the antitumour activity of ZD6126, a microtubule destabilising VDA, in combination with paclitaxel (PTX), a microtubule-stabilising cytotoxic drug, and the influence of schedule and sequence on the efficacy of the combination. Nude mice bearing MDA-MB-435 xenografts received weekly cycles of ZD6126 (200 mg kg(-1) i.p.) administered at different times before or after PTX (10, 20, and 40 mg kg(-1) i.v.). ZD6126 given 2 or 24 h after PTX showed no significant benefit, a result that was attributed to a protective effect of PTX against ZD6126-induced vascular damage and tumour necrosis, a hallmark of VDA activity. Paclitaxel counteracting activity was reduced by distancing drug administrations, and ZD6126 given 72 h after PTX potentiated the VDA's antitumour activity. Schedules with ZD6126 given before PTX improved therapeutic activity, which was paralleled by a VDA-induced increase in cell proliferation in the viable tumour tissue. Paclitaxel given 72 h after ZD6126 yielded the best response (50% tumours regressing). A single treatment with ZD6126 followed by weekly administration of PTX was sufficient to achieve a similar response (57% remissions). These findings show that schedule, sequence and timing are crucial in determining the antitumour efficacy of PTX in combination with ZD6126. Induction of tumour necrosis and increased proliferation in the remaining viable tumour tissue could be exploited as readouts to optimise schedules and maximise therapeutic efficacy.
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Inibidores da Angiogênese/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Compostos Organofosforados/uso terapêutico , Paclitaxel/uso terapêutico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Necrose , Neovascularização Patológica , Taxa de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated with the cytotoxic drug calicheamicin and approved for the treatment of relapsed acute myeloid leukaemia. As approximately 18% of acute lymphoblastic leukaemias (ALL) are also CD33 positive, we have investigated the cytotoxic activity of GO on CD33+ ALL cells in vitro and in vivo. 10 ng/ml GO induced 30-95% inhibition of thymidine uptake and 30-70% cell death in four freshly isolated and one in vivo passaged CD33+ ALL-cell cultures. Furthermore, an in vivo model of a CD33+ ALL carrying the Philadelphia chromosome [t(9;22)] was established. 5 x 10(6) ALL-2 cells inoculated in the tail vein of severe combined immunodeficient mice engrafted into haematopoietic organs, reaching a mean of 70%, 61% and 69% human CD45+ cells in bone marrow, spleen and liver, respectively, at 35 d. To test the therapeutic activity of GO, 50 or 100 microg immunotoxin was inoculated i.p. on days 7, 11 and 15 following tumour-cell inoculation. GO treatment dramatically inhibited expansion of ALL-2 cells in all tested organs and increased survival of tumour-injected animals by 28-41 d, relative to controls. These data demonstrated that GO is active both in vitro and in vivo against CD33+ ALL cells.
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Aminoglicosídeos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Imunotoxinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados , Morte Celular/efeitos dos fármacos , Feminino , Gemtuzumab , Humanos , Imunofenotipagem , Camundongos , Camundongos SCID , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The antineoplastic compound aplidine, a new marine-derived depsipeptide, has shown preclinical activity in vitro on haematological and solid tumour cell lines. It is currently in early phase clinical trials. The exact mechanism of action of this anticancer agent still needs to be clarified. We have previously reported that aplidine blocks the secretion of the angiogenic factor vascular endothelial growth factor (VEGF) by the human leukaemia cells MOLT-4, suggesting a possible effect on tumour angiogenesis. This study was designed to investigate the antiangiogenic effect of aplidine. In vivo, in the chick embryo allantoic membrane (CAM) assay, aplidine inhibited spontaneous angiogenesis, angiogenesis elicited by exogenous VEGF and FGF-2, and induced by VEGF overexpressing 1A9 ovarian carcinoma cells. In vitro, at concentrations achievable in the plasma of patients, aplidine inhibited endothelial cell functions related to angiogenesis. It affected VEGF- and FGF-2-induced endothelial cell proliferation, inhibited cell migration and invasiveness assessed in the Boyden chamber and blocked the production of matrix metalloproteinases (MMP-2 and MMP-9) by endothelial cells. Finally, aplidine prevented the formation of capillary-like structures by endothelial cells on Matrigel. These findings indicate that aplidine has antiangiogenic activity in vivo and inhibits endothelial cell functional responses to angiogenic stimuli in vitro. This effect might contribute to the antineoplastic activity of aplidine.
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Inibidores da Angiogênese/farmacologia , Depsipeptídeos , Neovascularização Patológica , Peptídeos Cíclicos/farmacologia , Animais , Bioensaio , Capilares , Técnicas de Cultura de Células , Embrião de Galinha , Células Endoteliais , Humanos , Metaloproteinases da Matriz/análise , Veias Umbilicais/citologiaRESUMO
The development of a functional vasculature within a tumour is a requisite for its growth and progression. This fact has led to the design of therapies directed toward the tumour vasculature, aiming either to prevent the formation of new vessels (anti-angiogenic) or to damage existing vessels (vascular targeting). The development of agents with different mechanisms of action requires powerful preclinical models for the analysis and optimization of these therapies. This review concerns 'classical' assays of angiogenesis in vitro and in vivo, recent approaches to target identification (analysis of gene and protein expression), and the study of morphological and functional changes in the vasculature in vivo (imaging techniques). It mainly describes assays designed for anti-angiogenic compounds, indicating, where possible, their application to the study of vascular-targeting agents.
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Inibidores da Angiogênese/uso terapêutico , Neoplasias/irrigação sanguínea , Animais , Apoptose , Divisão Celular , Avaliação de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Modelos Biológicos , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Peixe-ZebraRESUMO
Yondelis (trabectidin, ET-743) is a marine natural product that has shown activity both in preclinical systems and in human malignancies such as soft tissue sarcoma and ovarian cancers that are resistant to previous chemotherapies. Molecular pharmacological studies indicated that Yondelis interacts with DNA and DNA repair systems in a way that is different from Cisplatin (DDP). The current study was designed to investigate the effects of the combination of Yondelis and DDP in human cancer cell lines and in xenografts derived from different tumours. The in vitro studies performed in human TE-671 rhabdomyosarcoma, Igrov-1 and 1A9 human ovarian carcinoma cell lines showed additive effects or slight synergism. Several human tumour xenografts, such as TE-671 rhabdomyosarcoma, SK-N-DX neuroblastoma, FADU head and neck, LX-1 non-small cell lung cancer (NSCLC), H-187 melanoma and SKOV HOC 8 ovarian carcinoma, showed an antitumour effect for the combination that was greater than that of each drug when given as a single agent. No consistent changes in the activity were observed if Yondelis and DDP were given 1 h apart in sequence or simultaneously. An orthotopically transplanted human ovarian cancer HOC 8 growing in the peritoneal cavity of nude mice was used that is insensitive to Yondelis alone and only moderately sensitive to DDP alone. The combination of the two drugs produced a dramatic increase of survival lasting several months. In conclusion, the combination of Yondelis and DDP is synergistic in vivo (i.e. the antitumour effect is greater than that of each drug used as a single agent at the maximum tolerated dose (MTD)) in different human tumour xenografts. The two drugs can be combined at the MTD of each drug, thus indicating there are no overlapping toxicities. These results provide a rationale for testing the combination of Yondelis and DDP in the clinic.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Dioxóis/administração & dosagem , Dioxóis/efeitos adversos , Sinergismo Farmacológico , Feminino , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/efeitos adversos , Camundongos , Transplante de Neoplasias , Tetra-Hidroisoquinolinas , Trabectedina , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
We measured the levels of the vascular endothelial growth factor (VEGF), matrix metalloproteinases type 2 and type 9 (MMP-2 and MMP-9) and tissue inhibitors of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the plasma of patients with ovarian carcinoma (n=40), in other gynaecological pathologies (n=30) and in the plasma of healthy volunteers (n=26). MMP-2 and MMP-9 (pro and active forms) gelatinolytic activity was measured by zymography. Enzyme-linked immunosorbent assays (ELISA) were used to assay soluble VEGF and TIMPs. Preoperative plasma VEGF levels were significantly higher in patients with ovarian cancer than in healthy volunteers (P<0.0001) or patients with a benign gynaecological pathology (P<0.0001). The expression of pro-MMP-9 was higher in the plasma of ovarian cancer patients than in the plasma of women with non-malignant disease (P=0.01) or healthy women (P<0.0002). Pro-MMP-2 was detected in the plasma of ovarian cancer patients, but levels did not differ from those in non-malignant disease or healthy donor samples. Plasma TIMP-1 and TIMP-2 levels were significantly higher in patients with ovarian carcinomas than in healthy volunteers (P<0.0001 and P=0.006, respectively) or in the patients with a non-malignant pathology (P<0.0001 and P=0.002, respectively). Sub-group analysis showed that VEGF and pro-MMP-9 were higher in the plasma of patients with serous carcinomas than other histological types. Furthermore, plasma VEGF and pro-MMP-9 levels were higher in the plasma of cancer patients with thrombocytosis. Throughout the study, and in the univariate analysis, no correlation was found between the VEGF, MMP and TIMP levels. Only TIMP-1 was associated with a poor survival and mortality risk.
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Biomarcadores Tumorais/sangue , Neoplasias Ovarianas/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Fatores de Crescimento Endotelial/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The mechanism by which aplidine, a marine natural product in early clinical development as an anticancer agent, induces cell growth inhibition and apoptosis has been investigated in the human leukemia cell line MOLT-4. This cell line is characterized not only by the ability to secrete VEGF, but also for the presence on its surface of the VEGF receptor-1 (VEGFR-1). Previous studies from our laboratory concerned with evaluating early changes in gene expression induced by aplidine in MOLT-4 cells have shown that the drug decreases the expression of VEGFR-1 (Marchini et al. Proc Am Assoc Cancer Res 2000; 41: 833). Here, we report the ability of aplidine to block the VEGF/VEGFR-1 loop. We found that aplidine blocked VEGF secretion that was temporally followed by a decrease in both VEGF and VEGFR-1 production. Aplidine did not directly affect either VEGF transcription or stabilization of its mRNA. Transfection of MOLT-4 cells with an antisense VEGF cDNA construct, resulted in inhibition of colony formations. One clone, transfected with sense VEGF cDNA, secreting 8-10 times more VEGF than parental cells, was less sensitive to aplidine-induced cytotoxicity and apoptosis than control cells. Moreover, addition of VEGF in the medium decreased the activity of aplidine in MOLT-4 cells. These data demonstrate that aplidine inhibits the growth and induces apoptosis in MOLT-4 cells through the inhibition of VEGF secretion which blocks the VEGF/VEGFR-1 autocrine loop necessary for the growth of these cells.
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Antineoplásicos/farmacologia , Depsipeptídeos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Leucemia de Células T/tratamento farmacológico , Linfocinas/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Comunicação Autócrina , Divisão Celular/efeitos dos fármacos , Primers do DNA/química , Dactinomicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Meia-Vida , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Luciferases/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
Tumor neovascularization is controlled by a balance between positive and negative effectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These findings suggest a potential role of p73 in tumor angiogenesis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Neovascularização Patológica/genética , Proteínas Nucleares/fisiologia , Trombospondina 1/biossíntese , Animais , Northern Blotting , Carcinoma/patologia , Quimiotaxia/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Meios de Cultura Livres de Soro , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genes p53 , Humanos , Linfocinas/genética , Proteínas de Membrana , Camundongos , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Neoplasias Ovarianas/patologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombospondina 1/genética , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Paclitaxel resistance has been associated with overexpression of P-glycoprotein and alterations involving tubulin. To investigate the clinical relevance of these in vitro resistance mechanisms, we established 12 human ovarian carcinoma xenografts, using samples from patients before the start of therapy or after paclitaxel treatment. These xenografts showed a wide range of sensitivity to paclitaxel, and in 4 of them, very low levels of multidrug resistance-1 expression were detected. Using quantitative PCR and human specific primers, the expression of five beta-tubulin isotypes was determined. HM40 was the predominant, accounting for 84.7-98.7% of all tubulin; expression of the other four isotypes (Hbeta9, Hbeta4, H5beta, and Hbeta2) was also detected but at lower levels. No correlation could be demonstrated between isotype expression and paclitaxel sensitivity in these 12 xenografts. A similar pattern of beta-tubulin isotype expression was observed in a subset of cell lines from the National Cancer Institute-Anticancer Drug Screen. In these cell lines, however, a significant correlation between increased expression of Hbeta4 isotype and resistance to paclitaxel was found. Taken together, these results suggest that altered expression of specific beta-tubulin isotypes may not play a significant role in paclitaxel sensitivity in vivo and argue against a possible significance in a clinical setting.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/uso terapêutico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Distribuição Tecidual , Tubulina (Proteína)/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fibroblast growth factor-2 (FGF2) is a pleiotropic heparin-binding growth factor endowed with a potent angiogenic activity in vitro and in vivo. To investigate the impact of the modulation of FGF2 expression on the neovascularization at different stages of tumor growth, we generated stable transfectants (Tet-FGF2) from the human endometrial adenocarcinoma HEC-1-B cell line in which FGF2 expression is under the control of the tetracycline-responsive promoter (Tet-off system). After transfection, independent clones were obtained in which FGF2 mRNA and protein were up-regulated compared with parental cells. Also, the conditioned medium of Tet-FGF2 transfectants caused proliferation, urokinase-type plasminogen activator up-regulation, migration, and sprouting of cultured endothelial cells. A 3-day treatment of Tet-FGF2 cell cultures with tetracycline abolished FGF2 overexpression and the biological activity of the conditioned medium without affecting their proliferative capacity. Tet-FGF2 cells formed tumors when nude mice received s.c. injections. The administration of 2.0 mg/ml tetracycline in the drinking water before cell transplantation, continued throughout the whole experiment, inhibited FGF2 expression in Tet-FGF2 tumor lesions. This was paralleled by a significant decrease in the rate of tumor growth and vascularization to values similar to those observed in lesions generated by parental HEC-1-B cells. Tetracycline administration 20 days after tumor cell implant, although equally effective in reducing FGF2 expression and inhibiting tumor vascularity, only minimally impaired the growth of established Tet-FGF2 tumors. The results indicate that FGF2 expression deeply affects the initial tumor growth and neovascularization of HEC-1-B human endometrial adenocarcinoma in nude mice. On the contrary, the growth of established tumors appears to be independent of the inhibition of FGF2 expression and decreased vascular density. The possibility that a significant reduction of angiogenesis may not affect the progression of large tumors points to the use of antiangiogenic therapy in early tumor stage.
Assuntos
Adenocarcinoma/irrigação sanguínea , Neoplasias do Endométrio/irrigação sanguínea , Fator 2 de Crescimento de Fibroblastos/biossíntese , Neovascularização Patológica/metabolismo , Tetraciclina/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , DNA Complementar/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibitors of matrix metalloproteinases (MMPs), enzymes involved in the processes of tumor growth, angiogenesis, invasion and metastasis, represent a promising new potential approach to cancer therapy. Several synthetic inhibitors of MMPs have been developed, many of which are currently in clinical trials. This review will describe some inhibitors of MMPs, presenting results of preclinical studies and, where available, their current clinical status as well. Issues concerning the use of MMP inhibitors, the design of clinical trials and the assessment of clinical response will also be addressed.
Assuntos
Antineoplásicos/uso terapêutico , Metaloendopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Humanos , Invasividade Neoplásica/prevenção & controle , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológicoRESUMO
Inhibitors of proteases prevent tumor-associated matrix degradation, affecting tumor growth, angiogenesis and metastasis. Our study was designed to investigate the effect of inhibition of matrix metalloproteinases (MMPs) on the growth of experimental hemangiomas, using the model of murine endothelioma eEnd.1 cells. In nude mice, these cells generate hemangiomas, consisting mostly of host-recruited endothelial cells, whose growth requires the activity of MMPs. In vitro, eEnd.1 cells produce factors that recruit endothelial cells and stimulate them to release MMPs. Over-expression of TIMP-2, following retrovirus-mediated gene transfer, decreased tumor growth in vivo. The infected clone CR1, which produces high levels of TIMP-2 (as assessed by Northern blot, ELISA and reverse zymography), formed slow-growing tumors that did not grow beyond 0.4 g, while clone 1H, which produces little TIMP-2, grew not dissimilarly to mock-infected cells and parental e.End.1 cells. Histologically, control tumors presented the features of cavernous hemangiomas, while CR1 tumors had a more solid pattern, showing foci of apoptotic cells. In vitro, TIMP-2 over-expression had no autocrine anti-proliferative effect on endothelioma cells but reduced their ability to recruit endothelial cells. CR1 cells lacked the capacity of mock-infected or parental eEnd.1 cells to stimulate endothelial cell motility and invasiveness. Antibodies against TIMP-2 restored the ability of CR1 to induce endothelial cell invasion. We conclude that, in this model, genetic increase of TIMP-2 inhibits tumor growth, apparently by affecting the recruitment and organization of host endothelial cells by the transformed cells.