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1.
ACS Omega ; 9(3): 3781-3792, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38284048

RESUMO

Colloidosomes are microcapsules whose shells are composed of cumulated or fused colloidal particles. When colloidosomes are used for in situ encapsulation, it is still a challenge to achieve a high encapsulation efficiency and controllable release by an effective fabrication method. Herein, we present a highly efficient route for the large-scale preparation of colloidosomes. The biodegradable polylactic acid (PLA) nanoparticles (NPs) as shell materials can be synthesized using an antisolvent precipitation method, and the possible formation mechanism was given through the molecular dynamics (MD) simulation. The theoretical values are basically consistent with the experimental results. Through the use of the modified and unmodified PLA NPs, the colloidosomes with controllable shell porosities can be easily constructed using spray drying technology. We also investigate the mechanism of colloidosomes successfully self-assembled by PLA NPs with various factors of inlet temperature, feed rate, and flow rates of compressed air. Furthermore, avermectin (AVM) was used as a model for in situ encapsulation and a controllable release. The spherical modified colloidosomes encapsulating AVM not only achieve a small mean diameter of 1.57 µm but also realize a high encapsulation efficiency of 89.7% and impermeability, which can be further verified by the MD simulation. AVM molecules gather around and clog the shell pores during the evaporation of water molecules. More importantly, the PLA colloidosomes also reveal excellent UV-shielding properties, which can protect AVM from photodegradation.

2.
Int J Mol Sci ; 24(24)2023 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-38139113

RESUMO

The successful mating of the hoverfly and the search for prey aphids are of great significance for biological control and are usually mediated by chemical cues. The odorant receptor co-receptor (Orco) genes play a crucial role in the process of insect odor perception. However, the function of Orco in the mating and prey-seeking behaviors of the hoverfly remains relatively unexplored. In this study, we characterized the Orco gene from the hoverfly, Eupeodes corollae, a natural enemy insect. We used the CRISPR/Cas9 technique to knock out the Orco gene of E. corollae, and the EcorOrco-/- homozygous mutant was verified by the genotype analysis. Fluorescence in situ hybridization showed that the antennal ORN of EcorOrco-/- mutant lack Orco staining. Electroantennogram (EAG) results showed that the adult mutant almost lost the electrophysiological response to 15 odorants from three types. The two-way choice assay and the glass Y-tube olfactometer indicated that both the larvae and adults of hoverflies lost their behavioral preference to the aphid alarm pheromone (E)-ß-farnesene (EBF). In addition, the mating assay results showed a significant decrease in the mating rate of males following the knock out of the EcorOrco gene. Although the mating of females was not affected, the amount of eggs being laid and the hatching rate of the eggs were significantly reduced. These results indicated that the EcorOrco gene was not only involved in the detection of semiochemicals in hoverflies but also plays a pivotal role in the development of eggs. In conclusion, our results expand the comprehension of the chemoreceptive mechanisms in the hoverflies and offers valuable insights for the advancement of more sophisticated pest management strategies.


Assuntos
Dípteros , Receptores Odorantes , Animais , Feminino , Masculino , Odorantes , Receptores Odorantes/genética , Hibridização in Situ Fluorescente , Dípteros/genética , Insetos/genética , Feromônios , Mutagênese , Proteínas de Insetos/genética
3.
J Agric Food Chem ; 71(4): 1837-1844, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36682010

RESUMO

Odorant receptors (ORs) in insects are crucial for the detection of chemical signals. However, the functions of the conserved OR genes among insect species are rarely studied. In this study, we analyzed a well-conserved OR clade in Diptera insects and cloned a gene from this clade, EcorOR4, in the hoverfly Eupeodes corollae. Real-time quantitative PCR showed that EcorOR4 was highly expressed in the antennae and upregulated in the mated females, and in vitro functional characterization showed that EcorOR4 was narrowly tuned to 1-octen-3-ol. Electroantennogram assays revealed that the antennal response of mated females to 1-octen-3-ol was significantly higher than that of mated males, but no significant differences were observed between male and female virgins. Finally, a Y-tube olfactometer bioassay showed that 1-octen-3-ol is an attractant for only mated female E. corollae adults. These results demonstrate that EcorOR4 is involved in the detection of 1-octen-3-ol and that this compound may affect the host-finding and oviposition behavior in female E. corollae.


Assuntos
Dípteros , Receptores Odorantes , Animais , Feminino , Masculino , Receptores Odorantes/genética , Dípteros/genética , Octanóis , Oviposição
4.
J Agric Food Chem ; 68(44): 12212-12220, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33103425

RESUMO

Flowering plants attract pollinators with volatile chemicals that include aromatic compounds. Syrphid flies are the largest group of flower visitors in Diptera, but little is known about how they detect floral scents at the molecular level. Here, electroantennogram (EAG) recordings from the antennae of Eupeodes corollae were used to measure responses from 14 aromatic compounds. To identify odorant receptors (ORs) of E. corollae tuned to aromatic volatiles, we analyzed functional profiles of Drosophila melanogaster odorant receptors (ORs), DmelOR46a and DmelOR71a, which are narrowly tuned to phenolic compounds and represent the orthologues of E. corollae OR25 and OR28, respectively. The two genes that are expressed in the antennae of both sexes were functionally characterized. EcorOR25 is narrowly tuned to several structurally related floral scent volatiles, including eugenol, p-cresol, and methyl eugenol. Finally, choice behavior assays showed that eugenol and methyl eugenol were attractants for both sexes of E. corollae adults. This study identified the odorant receptors used by E. corollae to detect aromatic volatiles, suggesting environmentally friendly strategies to attract these beneficial insects.


Assuntos
Dípteros/metabolismo , Flores/química , Proteínas de Insetos/metabolismo , Receptores Odorantes/metabolismo , Compostos Orgânicos Voláteis/química , Animais , Dípteros/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Flores/parasitologia , Proteínas de Insetos/genética , Odorantes/análise , Receptores Odorantes/genética , Olfato
5.
Cancer Lett ; 258(2): 291-300, 2007 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-17950526

RESUMO

Both the insulin-like growth factor-I receptor (IGF-IR) and cyclooxygenase-2 (COX-2) are frequently overexpressed in pancreatic cancer. We hypothesized that IGF-IR is directly involved in induction of COX-2 and sought to investigate signaling pathways mediating this effect. Pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative receptor (IGF-IR DN) construct or empty vector (pcDNA). Cells were stimulated with IGF-I to determine activated signaling intermediates and induction of COX-2. Signaling pathways mediating COX-2 induction were identified using signaling inhibitors. IGF-I up-regulated COX-2 selectively via the MAPK/(Erk-1/2) pathway. In addition, IGF-IR DN cells showed a marked decrease in constitutive COX-2 and a blunted response to IGF-I. Similarly, treatment with an anti-IGF-IR antibody effectively inhibited IGF-IR and MAPK/Erk activation and decreased COX-2 in parental cells. In conclusion, activation of IGF-IR mediates COX-2 expression in human pancreatic cancer cells.


Assuntos
Ciclo-Oxigenase 2/genética , Fator de Crescimento Insulin-Like I/farmacologia , Receptor IGF Tipo 1/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Substratos do Receptor de Insulina , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção
6.
Clin Cancer Res ; 13(16): 4704-12, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17699847

RESUMO

PURPOSE: Gastrointestinal neuroendocrine tumors (NET) are rare heterogeneous tumors that hypersecrete neuropeptides. The scarcity of good gastrointestinal NET models has limited the ability to study potential therapeutic agents. We describe and characterize the establishment of a human midgut carcinoid tumor cell line carcinoid tumor 2 (CNDT2). EXPERIMENTAL DESIGN: Tumor cells (CNDT2) were isolated from a liver metastasis from a patient with a primary ileal carcinoid. After 9 weeks in culture, the cells were plated in soft agar, and cells from a single colony were put back in culture (CNDT2.1). Those CNDT2.1 cells were injected s.c. into nude mice. Cells were isolated from a single resultant tumor (CNDT2.5), cultured, and characterized by electron microscopy, reverse transcription-PCR, serotonin enzyme immunoassay, Western blotting, and immunohistochemical analysis for NET markers and potential therapeutic targets. RESULTS: CNDT2 cells grew in monolayers in vitro, formed colonies in soft agar, and formed tumors in mice. Electron microscopy revealed round, pleomorphic, electron-dense neurosecretory granules characteristic of NETs. Tumor xenografts exhibited the appearance of NETs with small "salt-and-pepper" nuclei on H&E staining and chromogranin A, synaptophysin, and CD56 on immunohistochemical staining. CNDT2.5 cells produced serotonin and expressed insulin-like growth factor receptor-I, platelet-derived growth factor receptor-beta, vascular endothelial growth factor receptor-1, cMET, epidermal growth factor receptor, neuropilin-1, and somatostatin receptors 1 to 5. Cytogenetic analysis revealed the presence of deletions at 2p and 6q and numerous translocations. CONCLUSION: The establishment of this human midgut carcinoid tumor cell line may serve as a useful model system for studying cell biology and novel targeted agents in preclinical models.


Assuntos
Tumor Carcinoide/patologia , Neoplasias do Íleo/patologia , Animais , Tumor Carcinoide/genética , Tumor Carcinoide/metabolismo , Linhagem Celular Tumoral , Aberrações Cromossômicas , Feminino , Humanos , Neoplasias do Íleo/genética , Neoplasias do Íleo/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Receptores de Fatores de Crescimento do Endotélio Vascular/análise , Serotonina/biossíntese , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/análise
7.
J Clin Invest ; 117(8): 2114-22, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17641778

RESUMO

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.


Assuntos
Neoplasias Colorretais/metabolismo , Expressão Gênica , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/metabolismo , Pericitos/metabolismo , Fator de Crescimento Derivado de Plaquetas/biossíntese , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Becaplermina , Benzamidas , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Complementar , Humanos , Mesilato de Imatinib , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pericitos/patologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-sis , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transfecção
8.
Ann Surg Oncol ; 14(10): 2838-46, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17653802

RESUMO

BACKGROUND: Colorectal carcinomas (CRC) express high levels of insulin-like growth factor-I/II (IGF-I/II) and the receptor (IGF-IR). We hypothesized that selective inhibition of IGF-IR would inhibit hepatic growth of human CRC in mice. METHODS: Human CRC cells were treated in vitro with anti-IGF-IR monoclonal antibody (MoAB) with and without oxaliplatin to assess cytotoxicity. The effect of anti-IGF-IR MoAB on IGF-I-induced vascular endothelial growth factor (VEGF) production in human CRC cells was assessed by Northern blot and ELISA. We injected human CRC cells intrahepatically in nude mice, and then administered anti-IGF-IR MoAB with and without oxaliplatin. We delayed treatment in one group until large hepatic tumors were present. We assessed tumors for apoptosis, proliferation, and angiogenesis. RESULTS: Anti-IGF-IR MoAB and oxaliplatin inhibited CRC cell growth in vitro and combination treatment was even more effective. IGF-I stimulation of CRC cells resulted in significant upregulation of VEGF and this was completely inhibited by pretreatment with anti-IGF-IR MoAB. Anti-IGF-IR MoAB significantly inhibited hepatic growth of tumors in mice. Anti-IGF-IR MoAB plus oxaliplatin led to a significantly greater inhibition of tumor growth. Anti-IGF-IR MoAB plus oxaliplatin was just as effective at inhibiting growth of larger, more advanced liver tumors. Anti-IGF-IR MoAB, alone and in combination with oxaliplatin, led to a significant increase in tumor cell apoptosis, and a significant inhibition of tumor cell proliferation and angiogenesis. CONCLUSIONS: These findings suggest that IGF-IR is a potential target for therapy in patients with advanced CRC.


Assuntos
Anticorpos Monoclonais/farmacologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Neoplasias Hepáticas Experimentais/secundário , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Northern Blotting , Ensaio de Imunoadsorção Enzimática , Células HT29/efeitos dos fármacos , Células HT29/patologia , Células HT29/transplante , Humanos , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/patologia , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Mol Cancer Ther ; 5(7): 1676-82, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16891453

RESUMO

Pancreatic carcinoma cells overexpress the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and the hepatocyte growth factor (HGF) receptor, c-Met, which are both known to mediate tumor cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells and that IGF-I-mediated migration and invasion depend on c-Met. Migration and invasion assays were done with the human pancreatic cancer cell line L3.6pl treated with PBS, IGF-I, HGF, or IGF-I plus HGF. To determine if c-Met is necessary for IGF-IR-mediated migration and invasion, c-Met was down-regulated in L3.6pl cells via adenoviral infection with a c-Met ribozyme before IGF-I treatment. IGF-I and HGF increased cell migration and invasion. Furthermore, IGF-I plus HGF had a greater than additive effect on cell migration and invasion compared with either growth factor alone. Down-regulation of c-Met nearly completely inhibited IGF-I-mediated migration and invasion. Our findings suggest that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells. Furthermore, c-Met is required for both HGF- and IGF-I-mediated migration and invasion. Elucidation of the signaling pathways that contribute to tumor progression and metastasis should provide a foundation for the development of targeted therapies for pancreatic carcinoma.


Assuntos
Carcinoma/patologia , Movimento Celular , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor IGF Tipo 1/metabolismo , Carcinoma/metabolismo , Movimento Celular/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores
11.
Clin Cancer Res ; 12(14 Pt 1): 4147-53, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16857785

RESUMO

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In preliminary studies, we observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells undergo EMT. EXPERIMENTAL DESIGN: The human CRC cell lines KM12L4 and HT29 were exposed to increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular changes characteristic of EMT were determined by immunofluorescence staining and Western blot analyses. RESULTS: The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. KM12L4 and HT29 OxR cells exhibited an approximately 8- to 15-fold increase in migrating and invading cells, respectively (P < 0.005 for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and beta-catenin from their usual membrane-bound complex to the cytoplasm and nucleus, respectively. The OxR cells also had decreased expression of the epithelial adhesion molecules E-cadherin and plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated transcription factor nuclear factor kappaB. CONCLUSION: We hypothesize that induction of EMT may contribute to the decreased efficacy of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable improvements in chemotherapy for metastatic disease.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Epitélio/metabolismo , Mesoderma/metabolismo , Compostos Organoplatínicos/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Imuno-Histoquímica , Invasividade Neoplásica , Metástase Neoplásica , Oxaliplatina , Fuso Acromático
12.
Clin Cancer Res ; 12(8): 2628-33, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16638876

RESUMO

PURPOSE: Both nitric oxide (NO) and vascular endothelial growth factor (VEGF) mediate tumor vascular function. Because these molecules regulate one another's expression, we hypothesized that NO synthase (NOS) inhibition produces effects comparable to those of anti-VEGF therapy on human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: L3.6pl human pancreatic cancer cells were s.c. implanted in nude mice. On day 6, mice were randomized to receive (a) PBS (control), (b) DC101 [VEGF receptor 2 (VEGFR-2) antibody] by i.p. injection, (c) N-nitro-l-arginine (NNLA; NOS inhibitor) in the drinking water, or (d) both DC101 and NNLA. Mice were killed on day 20. RESULTS: DC101 and NNLA as single agents inhibited tumor growth by approximately 50% to 60% (P < 0.008 for both). Furthermore, combined therapy inhibited mean tumor growth by 89% (P < 0.008). Combined inhibition of VEGFR-2 and NOS also decreased mean vessel counts by 65% (P < 0.03) and vessel area by 80% versus controls (P < 0.001). In contrast to DC101 where vessel diameter was similar to control, NNLA decreased mean vessel diameter by 42% (P < 0.001). NNLA also led to a 54% (P < 0.03) decrease in tumor uptake of the perfusion marker Hoechst 33342 versus controls whereas DC101 decreased Hoechst 33342 staining by 43% (P < 0.03). The combination of inhibitors decreased perfusion by 73% (P < 0.03). CONCLUSIONS: Although VEGFR-2 can mediate NOS activity, the combination of VEGFR-2 and NOS inhibition significantly increased the antivascular effect over single agent therapy. The addition of NOS inhibition led to an even further alteration of tumor vessel morphology and vascular perfusion compared with VEGFR-2 blockade, suggesting that NO and VEGFR-2 have distinct but complementary effects on the tumor vasculature.


Assuntos
Anticorpos Monoclonais/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Neoplasias Pancreáticas/prevenção & controle , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Vasos Sanguíneos/química , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Óxido Nítrico Sintase/metabolismo , Nitroarginina/uso terapêutico , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Distribuição Aleatória
13.
Cancer Res ; 66(1): 46-51, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16397214

RESUMO

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.


Assuntos
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Mesoderma/metabolismo , Mesoderma/patologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator B de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/farmacologia
14.
Cytokine ; 32(5): 206-12, 2005 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-16289960

RESUMO

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.


Assuntos
Movimento Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Músculo Liso Vascular/fisiologia , Neuropilina-1/metabolismo , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Neuropilina-1/genética , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos
15.
Cancer Res ; 65(17): 7775-81, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16140945

RESUMO

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.


Assuntos
Movimento Celular/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Amilorida/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias Hepáticas Experimentais/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neovascularização Patológica , Neoplasias Pancreáticas/irrigação sanguínea , Proteínas Proto-Oncogênicas c-met/fisiologia , Receptor IGF Tipo 1/fisiologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/biossíntese
16.
Cancer ; 104(2): 427-38, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15952180

RESUMO

BACKGROUND: Vascular endothelial growth factor receptor-1 (VEGFR-1) is one of three receptor tyrosine kinases for VEGF, a key regulator of angiogenesis in cancer. Although VEGFRs initially were believed to be expressed exclusively on endothelial cells (ECs), recent studies have demonstrated the presence of VEGFR-1 on non-EC types. The authors hypothesized that VEGFR-1 is present and functional in pancreatic carcinoma cells, contributing to the malignant phenotype. METHODS: The authors assessed the expression of VEGFR-1 and its ligands in 11 pancreatic carcinoma cell lines by reverse-transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and/or Western blot analysis. The function of VEGFR-1 was evaluated by treating two representative cell lines with VEGF-B, a selective ligand for VEGFR-1, and/or a specific anti-VEGFR-1 antibody and assessing the effects on signaling, migration, invasion, and proliferation. RESULTS: All 11 pancreatic carcinoma cell lines expressed VEGFR-1 mRNA and protein, as well as the VEGFR-1 ligands VEGF-A and VEGF-B. Two representative cell lines (L3.6 and Panc-1) exhibited VEGF-B-induced mitogen-activated protein kinase signaling. A VEGFR-1 neutralizing antibody abrogated signaling, confirming that the ligand effect was mediated through VEGFR-1. VEGFR-1 stimulation by VEGF-A or VEGF-B was found to promote migration in both cell lines. Panc-1 cells also demonstrated enhanced Matrigel invasion after VEGFR-1 stimulation. VEGFR-1-dependent migration and invasion were blocked by the VEGFR-1 neutralizing antibody. VEGFR-1 activation did not appear to enhance cell proliferation. CONCLUSIONS: VEGFR-1 appears to be expressed ubiquitously in pancreatic carcinoma cell lines, in which it induces signaling and promotes migration and invasion. Overexpression of VEGF in tumors may activate tumor cells bearing VEGFR-1 via an autocrine pathway. Agents targeting VEGF or its receptors may have a dual inhibitory effect on tumor growth by suppressing both angiogenesis and tumor cell function.


Assuntos
Neoplasias Pancreáticas/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Western Blotting , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Células Tumorais Cultivadas , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia
17.
Clin Cancer Res ; 11(7): 2662-9, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15814647

RESUMO

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.


Assuntos
RNA Interferente Pequeno/genética , Sarcoma de Ewing/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antígenos de Neoplasias/metabolismo , Apoptose , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Plasmídeos/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/mortalidade , Taxa de Sobrevida , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Ann Surg Oncol ; 12(4): 273-81, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15827676

RESUMO

Tyrosine kinase receptors mediate many critical cellular functions that contribute to tumor progression and metastasis and thus are potential targets for molecular-based cancer therapy. As has been found for many receptor tyrosine kinases, RON (recepteur d'origine nantais) and its ligand, macrophage-stimulating protein, have recently been implicated in the progression and metastasis of tumors. In in vitro experiments using colon and breast cancer cell lines, overexpression of RON led to increased invasion and migration of cancer cells and prevented apoptosis and anoikis. In addition, transgenic mice engineered to overexpress RON in the lung epithelium developed multiple pulmonary tumors, suggesting a role for RON in tumorigenesis. In human cancer specimens, increased RON expression has been demonstrated in colon, breast, ovarian, and lung tumors. Therefore, therapies designed to inhibit RON activation may hinder critical tumor survival mechanisms and play a role in the treatment of advanced disease.


Assuntos
Transformação Celular Neoplásica/metabolismo , Metástase Neoplásica/fisiopatologia , Receptores Proteína Tirosina Quinases , Animais , Adesão Celular , Linhagem Celular Tumoral , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo
19.
Ann Surg ; 241(5): 748-56; discussion 756-8, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15849510

RESUMO

OBJECTIVE: To determine whether insulinlike growth factor-I (IGF-I) and hepatocyte growth factor (HGF) cooperate to induce migration and invasion of human colorectal carcinoma (CRC) cells and whether the effects of IGF-I and/or HGF are mediated through activation of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system, a central mediator of tumor-cell migration and invasion. SUMMARY BACKGROUND DATA: CRC cells must invade through the basement membrane of the colon and migrate to form metastases. CRC cells are known to overexpress IGF-I receptor (IGF-IR), c-Met, and uPAR, 3 cell-surface receptors known to mediate cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion in CRC cells and that this signaling is dependent on uPAR. METHODS: KM12L4 human CRC cells were treated with IGF-I, HGF, or IGF-I + HGF in transwell migration and invasion chambers; cells that had migrated or invaded were counted. To determine the role of c-Met in IGF-I-induced migration and invasion, c-Met was inhibited by infection of cells with an adenovirus containing a c-Met ribozyme; transwell assays were then repeated. To determine the role of the uPA/uPAR system in IGF-I-induced CRC cell migration and invasion, transwell assays were repeated after pretreating cells with the uPA inhibitor amiloride or with neutralizing antibodies to uPA and uPAR. RESULTS: IGF-I and HGF, alone or in combination, increased cell migration and invasion. The c-Met ribozyme inhibited IGF-I- and HGF-mediated migration and invasion, indicating that c-Met is essential for these processes. uPA and uPAR inhibition blocked IGF-I- and HGF-mediated migration and invasion, suggesting that uPAR is downstream of IGF/IGF-IR and HGF/c-Met in the signaling pathways that mediate cell migration and invasion. CONCLUSIONS: IGF-I and HGF cooperate to induce migration and invasion of CRC cells, and c-Met and uPA/uPAR are required for IGF-I-mediated migration and invasion. In our in vitro model of CRC migration and invasion, uPA and uPAR appear to be downstream of IGF-IR and c-Met and are required for migration and invasion. Elucidation of the pathways that contribute to tumor progression and metastasis should provide a foundation for the rational development and use of targeted therapies for CRC.


Assuntos
Movimento Celular/fisiologia , Neoplasias Colorretais/patologia , Fator de Crescimento Insulin-Like I/fisiologia , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-met/fisiologia , Receptores de Superfície Celular/fisiologia , Linhagem Celular Tumoral , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia
20.
Cancer ; 103(8): 1561-70, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15754332

RESUMO

It is believed that impairments in delivery of antineoplastic agents to solid tumors result from abnormalities of the tumor microenvironment. Vascular endothelial growth factor (VEGF), the prototypical angiogenic molecule, is one of the main factors responsible for the development and maintenance of the aberrant tumor vascular network, which is characterized by chaotic, leaky blood vessels with high interstitial fluid pressure and inefficient blood flow. The authors proposed that anti-VEGF therapy would reduce the elevated interstitial fluid pressure in tumors, thereby improving blood flow and potentially improving delivery of cytotoxic agents to tumor cells. For the current report, the authors reviewed characteristics of the abnormal tumor vasculature created under the influence of VEGF, the resulting tumor microenvironment, how the tumor microenvironment may impede delivery of antineoplastic agents, and how the combination of anti-VEGF and cytotoxic therapy may maximize the efficacy of antineoplastic treatment regimens.


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Humanos , Neoplasias/irrigação sanguínea
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