RESUMO
BACKGROUND: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. AIM: The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. METHODS/RESULTS: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. CONCLUSIONS: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.
Assuntos
Indóis/farmacologia , Indóis/uso terapêutico , Niacinamida/análogos & derivados , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Neuroendócrino , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/fisiologia , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The major cause of death from cancer is metastases that are resistant to conventional therapies. The resistance of metastatic tumor cells to chemotherapy can be caused by their intrinsic properties, such as increased expression of the mdr genes. PURPOSE: The purpose of our present study was to determine some of the mechanisms by which the organ microenvironment influences the response of tumor cells to chemotherapy. METHODS: Murine CT-26 colon cancer cells growing in continuous culture (parental cells) were harvested and injected subcutaneously into the lateral flank (to produce subcutaneous tumors) or the lateral tail vein (to produce experimental lung metastases) of 10 8-week-old syngeneic male BALB/c mice. Seven days after tumor-cell injection, the mice were given intravenous injections of either doxorubicin (10 mg/kg) or 0.9% NaCl (controls). This in vivo injection was repeated 7 days later. Mice with subcutaneous tumors and lung metastases were killed by cervical dislocation on day 21, and tumor samples from control mice were harvested and adapted to culture. The sensitivity of the cultured cells to doxorubicin and fluorouracil (5-FU) was determined at multiple time points. Levels of mdr-1 DNA were measured by slot-blot and Southern-blot analyses. mdr mRNA expression levels were measured by Northern-blot analysis using mdr-1- and mdr-3-specific hybridization probes, and P-glycoprotein level was determined by fluorescence-activated cell sorting using different monoclonal antibodies. RESULTS: Treatment with doxorubicin produced 80% growth inhibition of CT-26 subcutaneous tumors but had little effect on the number (and size) of experimental lung metastases. Collectively, the results suggest that the multidrug-resistant phenotype developed in CT-26 cells growing in the lung environment. Cultures established from lung metastases were initially resistant to doxorubicin (but not to 5-FU) and showed elevated expression of mdr-1 mRNA transcripts and P-glycoprotein. This resistance could be overcome by verapamil and disappeared after 21 days in culture. No mdr gene amplification was detected. The expression level of mdr-specific mRNA (predominance of mdr-1) and P-glycoprotein was directly associated with resistance to doxorubicin. CONCLUSIONS: Results of this study have demonstrated that the in vivo sensitivity of murine CT-26 colon carcinoma cells to doxorubicin depends on the organ environment. The organ environment can influence the P-glycoprotein-mediated multidrug-resistant phenotype in tumor cells, and the increased expression of P-glycoprotein is transient; once removed from the environment (lung), the cell's resistance reverts to that of the sensitive parent cells.
Assuntos
Carcinoma/tratamento farmacológico , Proteínas de Transporte/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Resistência a Medicamentos , Glicoproteínas de Membrana/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Carcinoma/genética , Doxorrubicina/administração & dosagem , Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , RNA Mensageiro/genética , RNA Neoplásico/genética , Neoplasias Cutâneas/tratamento farmacológico , Verapamil/administração & dosagemRESUMO
BACKGROUND: Epidermal growth factor receptor (EGF-R) proteins are highly expressed in many tumors, including those of the cervix. We have observed previously that the introduction of a transcription unit containing an antisense sequence for the E6/E7 genes of human papillomavirus (HPV) 18, along with a transcription unit containing a sense complementary DNA sequence for the wild-type retinoblastoma (Rb) gene, decreased the growth of human cervical carcinoma HeLa cells (HPV 18 positive) both in vitro and in vivo. To clarify the regulatory mechanisms by which this reduction in cell proliferation occurred, we studied the expression of EGF-R proteins in these cells. METHODS: Western blot and northern blot techniques were used to measure EGF-R expression, and a pulse-chase immunoprecipitation assay was used to measure the stability of EGF-R protein in HeLa cells and HeLa cells that had been transfected with the antisense E6/E7 or sense Rb sequences. Cell proliferation was measured by use of a tetrazolium-based colorimetric assay for numbers of viable cells. RESULTS: The introduction of sense Rb or antisense E6/E7 transcription units or a combination of these two transcription units into HeLa cells dramatically decreased the level of EGF-R proteins in these cells; EGF-R levels were not affected at the transcriptional level but at the post-transcriptional level. Addition of the anti-EGF-R-specific monoclonal antibody 225mAb to HeLa cells caused 53% (95% confidence interval = 44%-62%) growth inhibition. CONCLUSIONS: These results suggest that HeLa cervical carcinoma cells are dependent on EGF-R for proliferation and that changes in functional levels of the E6/E7 HPV proteins and endogenous Rb proteins may alter the growth rate of cervical cancer cell lines by reducing the stability of EGF-R at the post-transcriptional level.
Assuntos
Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes do Retinoblastoma/genética , Oligonucleotídeos Antissenso/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genética , RNA Antissenso/metabolismo , Proteínas Repressoras , Northern Blotting , Western Blotting , Células HeLa , Humanos , RNA Mensageiro/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Single-cell clones from primary and lung metastatic tumors have been evaluated for the state of the viral-Kirsten-ras oncogene (v-Ki-ras) by Southern blot analysis after injection of Kirsten sarcoma virus-transformed BALB/c 3T3 cells (KiMSV, with a replication-defective provirus) into athymic nude mice by four different injection routes. While all clones of early-passage KiMSV cells contained an EcoRI-generated 5.3-kilobase DNA fragment at high dosage level, most clones of late-passage cells had lost this v-Ki-ras fragment or had greatly diminished levels. However, all clones of all tumors (greater than 90 tested) obtained after injection of these late-passage cells contained a dosage of the 5.3-kilobase v-Ki-ras band similar to that of the early-passage KiMSV cells, suggesting either a very strong selection for v-Ki-ras-bearing cells of the early-passage type in tumor formation and/or the ability of a subset of late-passage cells to amplify this gene to some minimal dosage level. Both flow cytometric analyses for DNA content and quantitation of chromosomes showed that all primary and lung metastatic tumors had more than twice the number of chromosomes as the late-passage KiMSV cells; however, four of 80 late-passage cells had a chromosome count in the range of tumors, consistent with their importance in tumor generation and possibly amplification of the v-Ki-ras-bearing chromosome. Clonal analyses of lung micrometastatic tumors revealed a v-Ki-ras blot pattern identical to that of the s.c. primary tumors. However, two of five lung metastases from the footpad (as large rapidly growing nodules) and i.v. routes had multiple copies of v-Ki-ras in new sites; a second injection round led to even greater complexity in v-Ki-ras patterns in clones of lung tumors. Two assays were used to demonstrate that these new v-Ki-ras integrations were generated by superinfection with a "helper" retrovirus, not sarcomagenic by itself in the nude mice, that led to rescue/reinfection of tumor cells with the defective Kirsten sarcoma proviral genome--cellular transformation of 3T3 or C3H10T1/2 cells and RNA dot blot analyses for medium-secreted retrovirus specific for LTR or v-Ki-ras sequences. This "helper" retrovirus could not be detected in early- or late-passage KiMSV cells used for inoculation but could be detected in certain tissues of normal nude mice, demonstrating its in vivo origin.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Amplificação de Genes , Neoplasias Experimentais/genética , Oncogenes , Recombinação Genética , Animais , DNA/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Sequências Repetitivas de Ácido Nucleico , Retroviridae/genética , Ativação ViralRESUMO
The purpose of this study was to determine whether nm23 steady-state mRNA expression levels correlate with metastatic potential of mouse K-1735 melanoma cells, human KM12 colon cancer cells, and human SN12 renal cancer cells. Since neoplasms are heterogeneous and contain subpopulations of cells with different metastatic potentials, we analyzed multiple sets of nonmetastatic and metastatic clones isolated from each neoplasm. In addition, we also examined nine somatic cell hybrids produced by the fusion of nonmetastatic and metastatic K-1735 clones. In the mouse melanoma, we found heterogeneity in nm23-1 steady-state expression levels among the clones and hybrids that did not correlate with their metastatic phenotype. Clones isolated from human colon or renal carcinomas expressed similar levels of nm23-HI regardless of metastatic potential in nude mice. All of the human tumor cells were heterozygous for the nm23-HI-specific allelic DNA fragments, with no allelic deletions or gross alterations detected. Since the failure of tumor cells to produce metastasis can be due to multiple deficiencies, these data stress the importance of using independent clones with different metastatic potentials for the analysis of gene regulation of this process.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , Núcleosídeo-Difosfato Quinase , Proteínas/genética , Fatores de Transcrição , Alelos , Animais , Células Clonais/metabolismo , Neoplasias do Colo/genética , Deleção de Genes , Humanos , Células Híbridas/metabolismo , Neoplasias Renais/genética , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C3H , Nucleosídeo NM23 Difosfato Quinases , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismoRESUMO
Fas/APO-1 is a cell surface protein known to trigger apoptosis in a variety of cell types upon specific antibody binding. Although extensively studied on normal and malignant hematopoietic cells, little is known about Fas/APO-1 on nonhematopoietic cells. In the study presented here, we have examined Fas/APO-1 expression and function on 11 human tumors of nonhematopoietic origin. By flow cytometric analysis, Fas/APO-1 was expressed on 10 of the 11 tumors at levels comparable to those previously reported for lymphoid cells sensitive to the cytolytic effects of anti-Fas. Despite abundant cell surface expression, only 4 of the 10 Fas-positive tumors were sensitive to the cell-killing effects of anti-Fas. Moreover, anti-Fas enhanced the growth of 2 of 10 Fas-positive tumors. Additional studies using cycloheximide demonstrated that de novo protein synthesis was required for anti-Fas-triggered growth stimulation and, at least in one case, was responsible for the resistance to antibody-induced apoptosis. The biological effects initiated by anti-Fas engagement, however, were not correlated with endogenous bcl-2 expression. This report documents that: (a) Fas/APO-1 is widely expressed on cultured nonhematopoietic tumors; (b) the inherent susceptibility to anti-Fas-induced apoptosis is not correlated with expression of the Fas/APO-1 protein; (c) Fas/APO-1 engagement can result in growth enhancement; and (d) protective/growth-promoting proteins other than bcl-2 may contribute to the diverse spectrum of biological effects induced by anti-Fas engagement of the Fas/APO-1 protein.
Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos de Superfície/metabolismo , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Humanos , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas c-bcl-2 , Estimulação Química , Células Tumorais Cultivadas , Receptor fasRESUMO
Adhesion has been evaluated for tumor cell populations derived from Kirsten murine sarcoma virus (KiMSV)-transformed BALB/c 3T3 cells responding to substrata coated with intact plasma fibronectin (pFN), a family of related proteolytic fragments from pFN or cellular fibronectins (FNs), and the heparan sulfate-binding platelet factor-4 (PF4). Both early-passage KiMSV cells, harboring the viral Kirsten ras oncogene (v-Ki-ras+), and late-passage KiMSV cells, in which most cells have lost the oncogene (v-Ki-ras-), are compared with primary tumor and lung metastatic tumor cells after three routes of injection into nude mice; nontumorigenic v-Ki-ras- revertant cells have been cloned from the late-passage KiMSV population. Attachment of early-passage KiMSV, primary tumor, and lung metastatic tumor cells was optimal and resistant to soluble RGDS peptide in the medium on intact pFN, on fragment F-155 from pFN containing the RGDS cell-binding domain and the heparinII domain, and on PF4 but decreased for metastatic cells on F110 containing only the RGDS domain (and sensitive to RGDS peptide). Cytoplasmic spreading of early-passage KiMSV and all tumor cells was good to excellent in polygonal patterns on pFN and on F155, while most cells remained round on F110. Responses for KiMSV and tumor cells varied on different heparin-binding proteins; cells remained rounded or detached on F38 derived from pFN or on PF4 but spread effectively with long linear process extension on cellular FN-derived fragments F44 + 47 harboring the extra domaina sequence. That F44 + 47 may contain a new cell-binding site for v-Ki-ras+ cells was also indicated by resistance to bacterial heparitanase in cell responses on F44 + 47 but not on PF4 and extensive catabolism of proteoglycans in the substratum-attached material of these cells. v-Ki-ras- revertant cells, nontumorigenic in nude mice, have reacquired 3T3-like responses to proteolytic fragments, including much more effective spreading on PF4 or on F38 substrata, and have reverted in generating microfilament stress fibers on pFN, a competence lacking in all v-Ki-ras+ cells. These results indicate that (a) v-Ki-ras+ primary and metastatic tumor cells respond similarly to most proteolytic fragments of FNs harboring known binding domains, with a few exceptions; (b) v-Ki-ras gene expression correlates with a new cell surface receptor activity recognized by extra domaina-containing fragments from cellular FNs; and (c) loss of the viral oncogene to generate v-Ki-ras- revertant cells reverts their FN-mediated adhesion responses.
Assuntos
Adesão Celular , Transformação Celular Neoplásica , Genes ras , Vírus do Sarcoma Murino de Kirsten/genética , Vírus do Sarcoma Murino/genética , Actinas/ultraestrutura , Animais , Células Cultivadas , Células Clonais , Replicação do DNA , Fibronectinas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/microbiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica de Varredura , Fragmentos de Peptídeos , Peptídeo Hidrolases , RNA Mensageiro/análise , RNA Mensageiro/genética , Sarcoma Experimental/genética , Sarcoma Experimental/microbiologia , Sarcoma Experimental/patologia , Sarcoma Experimental/ultraestrutura , Células Tumorais Cultivadas/ultraestruturaRESUMO
We correlated the steady state transcription and protein secretion of interleukin 8 (IL-8) in 13 different human melanoma cell lines with their ability to grow and produce metastasis in nude mice. Highly metastatic cells expressed higher steady state levels of IL-8 mRNA transcripts than did low metastatic cells. In situ mRNA hybridization analyses confirmed the pattern of mRNA expression on a cellular level. Increased mRNA expression directly correlated with secretion of IL-8 protein as determined by enzyme-linked immunosorbent assay. Recombinant IL-8 stimulated the proliferation of low metastatic A375P cells in a dose-dependent manner, a stimulation that was abrogated by the use of a polyclonal antibody against IL-8. The data suggest that IL-8 can be an autocrine growth factor for human melanoma cells and that IL-8 is involved in melanoma metastasis.
Assuntos
Interleucina-8/fisiologia , Neoplasias Pulmonares/secundário , Melanoma/genética , Melanoma/secundário , Animais , Sequência de Bases , Northern Blotting , Divisão Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Hibridização In Situ , Interleucina-8/biossíntese , Interleucina-8/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , RNA Mensageiro/análise , RNA Mensageiro/genética , Estimulação Química , Transcrição GênicaRESUMO
Metastatic K-1735 murine melanoma cells are amelanotic in culture or in the subcutis of syngeneic mice. When injected into the internal carotid artery, these cells produce melanotic brain metastases. The production of melanin in tumor cells growing in the brain was directly correlated with induction of melanocyte-stimulating hormone receptor (MSH-R) steady-state mRNA transcripts. K-1735 cells isolated from brain lesions and implanted into the subcutis or grown in culture lose MSH-R transcripts and become amelanotic. In contrast to K-1735 cells, B16-BL6 melanoma cells constitutively produce melanin and express high levels of MSH-R mRNA regardless of the site of growth. Somatic cell hybrids between K-1735 and B16 cells produced melanin and expressed high levels of MSH-R mRNA transcripts, regardless of the site of growth, suggesting the dominance of the B16 phenotype. Treatment with alpha-MSH failed to upregulate MSH-R expression in cultured K-1735 cells or to maintain MSH-R expression in K-1735 cells isolated from brain metastases to be grown in culture. Responsiveness to alpha-MSH as determined by cell proliferation, melanin production, and intracellular accumulation of cyclic AMP directly correlated with MSH-R expression. These data demonstrate that a specific organ environment influences the phenotype of metastatic cells by regulation of specific genes that encode for cell surface receptors.
Assuntos
Neoplasias Encefálicas/secundário , Regulação Neoplásica da Expressão Gênica , Hormônios Estimuladores de Melanócitos/metabolismo , Melanoma/genética , Receptores do Hormônio Hipofisário/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Feminino , Melaninas/biossíntese , Camundongos , Camundongos Endogâmicos , Monofenol Mono-Oxigenase/metabolismo , Transplante de Neoplasias , RNA Mensageiro/análise , Receptores do Hormônio Hipofisário/biossíntese , Transcrição Gênica , Células Tumorais Cultivadas , alfa-MSH/farmacologiaRESUMO
To determine the effect of cell density on vascular endothelial growth factor (VEGF) expression and the mechanism of this effect, four human colon cancer cell lines were grown as sparse or confluent monolayers or as spheroids. VEGF mRNA increased > 2-fold in cells grown as confluent monolayers or spheroids compared with cells grown as sparse monolayers. Semiquantitative reverse transcription-PCR demonstrated a 2-fold increase in the larger VEGF mRNA isoform (189 bp) in confluent cells. Sparse cells grown in conditioned medium from confluent cells demonstrated a > 2-fold increase in VEGF mRNA. These data suggest that VEGF expression may be regulated by an unidentified soluble factor.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Sequência de Bases , Contagem de Células , Neoplasias do Colo/genética , Meios de Cultivo Condicionados , Fatores de Crescimento Endotelial/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Isomerismo , Linfocinas/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Interleukin 8 (IL-8) is mitogenic and chemotactic for endothelial cells. Within a neoplasm, IL-8 is secreted by inflammatory and neoplastic cells. The highly tumorigenic and highly metastatic human transitional cell carcinoma (TCC) cell line 253J B-V overexpresses IL-8 relative to the nontumorigenic and nometastatic 253J-P cell line. To determine whether IL-8 expression regulates tumorigenicity and metastasis in human TCC, 253J B-V cells were transfected with the full-sequence antisense (AS) cDNA for IL-8, whereas 253J-P cells were transfected with the full-length IL-8 cDNA, and control cells for each were transfected with the neomycin resistance (Neo) gene. In vitro, sense-transfected 253J-P cells overexpressed IL-8-specific mRNA and protein, whereas both of these were markedly reduced in AS-IL-8-transfected 253J B-V cells relative to controls. Moreover, sense-transfected cells showed up-regulation in matrix metalloproteinase type 9 mRNA, collagenase activity, and increased invasiveness through Matrigel-coated filters, whereas these measures were lower in AS-transfected cells relative to controls. After implantation into the bladders of athymic nude mice, the sense-transfected 253J-P cells acquired increased tumorigenicity and metastasis, whereas the AS-transfected cells significantly inhibited tumorigenicity and metastases in the 253J B-V cell lines. This effect was accompanied by reduced IL-8 expression and microvessel density. These studies demonstrate that IL-8 expression enhances angiogenic activity through the induction of matrix metalloproteinase type 9 and subsequently regulates the tumorigenesis and production of spontaneous metastases of human TCC.
Assuntos
Carcinoma de Células de Transição/patologia , Interleucina-8/metabolismo , Metástase Linfática , Neovascularização Patológica , Neoplasias da Bexiga Urinária/patologia , Animais , Carcinoma de Células de Transição/irrigação sanguínea , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/secundário , Colágeno/metabolismo , Colagenases/metabolismo , Combinação de Medicamentos , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-8/genética , Laminina/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Regiões Promotoras Genéticas/genética , Proteoglicanas/metabolismo , Estabilidade de RNA , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/irrigação sanguínea , Neoplasias da Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We determined whether down-regulation of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of a novel EGF-R tyrosine kinase inhibitor (PKI166) alone or in combination with gemcitabine (administered i.p.) can inhibit growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Therapy beginning 7 days after orthotopic injection of L3.6pl human pancreatic cancer cells reduced the volume of pancreatic tumors by 59% in mice treated with gemcitabine only, by 45% in those treated with PKI166 only, and by 85% in those given both drugs. The combination therapy also significantly inhibited lymph node and liver metastasis, which led to a significant increase in overall survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with significant reduction in tumor cell production of VEGF and IL-8, which in turn correlated with a significant decrease in microvessel density and an increase in apoptotic endothelial cells. Collectively, our results demonstrate that oral administration of an EGF-R tyrosine kinase inhibitor decreased growth and metastasis of human pancreatic cancer growing orthotopically in nude mice and increased survival. The therapeutic effects were mediated in part by inhibition of tumor-induced angiogenesis attributable to a decrease in production of proangiogenic molecules by tumor cells and increased apoptosis of tumor-associated endothelial cells.
Assuntos
Apoptose/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Administração Oral , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Western Blotting , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Regulação para Baixo , Endotélio/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , GencitabinaRESUMO
We have developed a rapid colorimetric in situ mRNA hybridization procedure to analyze epidermal growth factor receptor (EGF-R) transcripts in paraffin-embedded surgical specimens of human colon carcinomas. This technique is based on the use of 24-base oligonucleotide probes labeled with 6 biotin molecules at the 3' end. mRNA integrity was verified using a hyperbiotinylated 30-residue-long deoxythymidylate oligonucleotide probe, and the specificity of the reaction was confirmed by using labeled EGF-R-specific sense and antisense probes. Avidin alkaline phosphatase detection and the capillary technology used in the Microprobe System allowed for completion of the procedure in under 5 h. The human A431 epidermoid carcinoma cells growing in culture and fixed with formalin as well as paraffin-embedded sections of this tumor growing s.c. in nude mice served as positive controls. In situ hybridization with antisense EGF-R oligonucleotide probes directly correlated with EGF-R mRNA and protein levels observed by Northern blot and immunohistochemistry, respectively. In situ hybridization of paraffin-embedded sections of primary human colon carcinoma and metastases from liver and lymph node revealed cell-specific staining with EGF-R antisense oligonucleotide probes that correlated directly with Northern blot and immunohistochemistry analyses. Since this rapid and sensitive in situ mRNA hybridization technique can be used in properly preserved paraffin-embedded tissue, it allows for retrospective analyses of human tumor specimens using archival material.
Assuntos
Carcinoma/química , Neoplasias do Colo/química , Receptores ErbB/genética , Hibridização In Situ/métodos , RNA Mensageiro/análise , Animais , Sequência de Bases , Northern Blotting , Carcinoma de Células Escamosas/química , Colorimetria , Receptores ErbB/análise , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Human SAOS-2 osteogenic sarcoma cells are not metastatic in nude mice and do not express p53. We have selected a variant line (SAOS-LM2) that is tumorigenic and metastatic in nude mice. These cells were transfected with the p53 wild-type (p53wt) or mutated (p53mut 143A) gene, whose expression was verified by reverse transcriptase PCR, cDNA sequencing, and protein immunoprecipitation. Cells were injected i.v. into nude mice, and 4 months later, the mice were necropsied. All cell lines produced a similar number of visible lung metastases, albeit of different sizes. Microscopic examination revealed that most lung metastases in mice injected with p53wt cells (but not p53mut 143A or control cells) consisted of osteoid matrix and apoptotic cells. Expression of either p53wt or p53mut 143A verified the origin of the metastases. These data suggest that transfection of SAOS-LM2 cells with p53wt is associated with in vivo induction of terminal differentiation and apoptosis that inhibit progressive growth of metastases.
Assuntos
Apoptose/genética , Neoplasias Ósseas/patologia , Genes p53 , Neoplasias Pulmonares/patologia , Osteossarcoma/patologia , Animais , Sequência de Bases , Neoplasias Ósseas/genética , Diferenciação Celular , Primers do DNA , Humanos , Hibridização In Situ , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Osteossarcoma/genética , Osteossarcoma/secundário , Transfecção , Células Tumorais CultivadasRESUMO
The purpose of this study was to determine whether production of liver metastasis by human colon carcinoma (HCC) cells depends on the response of tumor cells to organ-derived growth factors. HCC cells were isolated from several surgical specimens whose malignant potential differed (Dukes' stage B or D tumors), adapted to grow in culture, and assessed for expression of the epidermal growth factor receptor (EGF-R). Northern blot analyses revealed that highly metastatic HCC cells expressed >5-fold the number of EGF-R mRNA transcripts as low metastatic cells. The level of mRNA correlated with the amount of EGF-R protein as detected by Western blotting, immunohistochemistry, and Scatchard analyses. HCC growth response in vitro to picograms of transforming growth factor alpha was associated with functional cell surface EGF-Rs as determined by receptor tyrosine kinase activity assays. The EGF-R gene was not amplified or rearranged in highly metastatic cells. However, fluorescence in situ hybridization analysis showed that the copy number of chromosome 7 was higher in the highly metastatic cells. HCC cells were selected in vitro for low or high expression of EGF-R. Subsequent to injection into nude mice, only cells with high expression of EGF-R produced a high incidence of liver metastasis. These data demonstrate that expression of EGF-R by HCC cells directly correlates with their ability to produce hepatic metastasis.
Assuntos
Neoplasias do Colo/patologia , Receptores ErbB/genética , Neoplasias Hepáticas/secundário , Metástase Neoplásica , Transcrição Gênica , Animais , Carcinoma de Células Escamosas , Divisão Celular/efeitos dos fármacos , Mapeamento Cromossômico , Cromossomos Humanos Par 7 , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Receptores ErbB/análise , Receptores ErbB/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , RNA Mensageiro/análise , Fator de Crescimento Transformador alfa/farmacologia , Fator de Crescimento Transformador alfa/fisiologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Epidermal growth factor receptor (EGF-R), a transmembrane glycoprotein that mediates the mitogenic response of cells to epidermal growth factor, is highly expressed on malignant human bladder cancer cells. The 4,5-dianilinophthalimides represent a novel class of inhibitors of the EGF-R family of tyrosine kinase with selectivity at the enzymatic and cellular levels. Two compounds of this class, CGP 54211 and CGP 53353, inhibited tyrosine kinase activity of the EGF-R in five different human transitional cell carcinoma lines. The compounds also produced cytostasis in vitro. Highly metastatic human 253J B-V cells were implanted in the bladder wall of nude mice. The daily oral administration of CGP 54211 inhibited the level of EGF-R phosphorylation in this tumor; necrosis and inhibition of tumor growth paralleled this inhibition.
Assuntos
Carcinoma de Células de Transição/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Ftalimidas/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Administração Oral , Animais , Carcinoma de Células de Transição/enzimologia , Carcinoma de Células de Transição/patologia , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Receptores ErbB/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Ftalimidas/farmacologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Previously we reported that when cells from the human transitional cell carcinoma cell line 253J B-V growing orthotopically within the bladder of athymic nude mice were treated with the anti-epidermal growth factor receptor monoclonal antibody C225, angiogenesis was inhibited, resulting in regression of the primary tumor and inhibition of metastasis. In this study, we evaluated whether paclitaxel enhanced this therapeutic effect of C225. In vitro, the proliferation of 253J B-V cells was inhibited more by the combination of C225 and paclitaxel than with either agent alone. In vivo therapy with C225 and paclitaxel resulted in significantly greater regression of tumors compared with either agent alone. Median bladder tumor weight was 85 mg (range, 69-133 mg) compared with 168 mg (range, 72-288 mg) after C225 alone (P < 0.05), and 273 mg (range, 83-563 mg) after paclitaxel alone (P < 0.005). The incidence of spontaneous lymph node metastasis was also reduced by the combination of C225 with paclitaxel, although this result did not significantly differ from results after the use of C225 alone. Treatment with paclitaxel and C225 down-regulated the expression of basic fibroblast growth factor, vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase type 9 and inhibited tumor-induced neovascularity compared with untreated controls (P < 0.005). Moreover, the combination of C225 and paclitaxel enhanced apoptosis in tumor and endothelial cells compared with either agent alone (P < 0.005). These studies indicate that therapy with paclitaxel increases the ability of C225 to inhibit tumorigenicity and metastasis. This effect is mediated by inhibition of angiogenesis and induction of apoptosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/imunologia , Carcinoma de Células de Transição/terapia , Receptores ErbB/antagonistas & inibidores , Paclitaxel/uso terapêutico , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Animais , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Divisão Celular , Cetuximab , Terapia Combinada , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Regulação para Baixo , Fatores de Crescimento Endotelial/biossíntese , Endotélio/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Concentração Inibidora 50 , Interleucina-8/biossíntese , Metástase Linfática , Linfocinas/biossíntese , Masculino , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Tamanho do Órgão/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial cell growth factor (VEGF) regulates angiogenesis and metastasis of bladder cancer (transitional cell carcinoma, TCC) through binding to VEGF receptor-2 (VEGFR-2). In this study, we evaluated whether the anti-VEGFR monoclonal antibody (Mab) DC101 in combination with paclitaxel inhibited tumorigenesis, angiogenesis, and metastasis of human TCC growing within the bladder of athymic nude mice. In vivo therapy with Mab DC101 and paclitaxel induced significant regression of bladder tumors compared with either agent alone. Median bladder weights were reduced from 601 mg in untreated controls, 422 mg in mice treated with paclitaxel alone (P < 0.005), 361 mg in mice treated with DC101 alone (P < 0.005), and 113 mg in mice that received combination therapy (P < 0.0005). Only one of nine mice developed spontaneous lymph node metastasis after combined treatment, compared with seven of seven untreated controls (P < 0.0005), six of eight after DC101 (P < 0.01), and five of eight mice after paclitaxel (P < 0.05). Combined treatment with both paclitaxel and DC101 inhibited tumor-induced neovascularity compared with all other groups (P < 0.005), without altering the expression of VEGF or flk1. Mab DC101 and paclitaxel combined enhanced apoptosis in the tumor and endothelial cells compared with other treatment (P < 0.005). These studies indicate that Mab DC101, which blocks VEGFR-2 function, has significant efficacy against human TCC, especially when combined with the chemotherapeutic agent paclitaxel. The antitumor effect was mediated by inhibition of angiogenesis and induction of both tumor cell and endothelial cell apoptosis.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Paclitaxel/uso terapêutico , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Apoptose , Carcinoma de Células de Transição/irrigação sanguínea , Carcinoma de Células de Transição/patologia , Divisão Celular , Fatores de Crescimento Endotelial/genética , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Interleucina-8/genética , Linfocinas/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Nus , Microcirculação/patologia , Neovascularização Patológica/prevenção & controle , RNA Mensageiro/análise , Receptores de Fatores de Crescimento do Endotélio Vascular , Transcrição Gênica , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/irrigação sanguínea , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epidermal growth factor receptor (EGFR) regulates the growth and progression of human transitional cell carcinoma (TCC) of the bladder. We have shown that therapy targeting EGFR inhibited the growth of human TCC established orthotopically in nude mice. The purpose of this study was to evaluate whether EGFR-directed therapy affects angiogenesis associated with the growth and metastasis of human TCC. We determined the cytostatic effect and the effect on production of angiogenic factors after in vitro treatment of the human TCC cell line 253J B-V with MAb C225, a chimerized monoclonal anti-EGFR antibody. The 253J B-V cells were implanted orthotopically into athymic nude mice, and established tumors (4 weeks) were treated with i.p. MAb C225. Expression of the angiogenic factors vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) was evaluated by immunohistochemistry and in situ mRNA hybridization analyses and correlated with microvessel density evaluated after immunohistochemical staining with anti-CD31. In vitro treatment with MAb C225 inhibited mRNA and protein production of VEGF, IL-8, and bFGF by 253J B-V cells in a dose-dependent manner. MAb C225 therapy of nude mice with established TCCs growing orthotopically resulted in inhibition of growth and metastasis compared with controls (P <0.0005). VEGF, IL-8, and bFGF expression was significantly lower in treated tumors than in controls. The down-regulation of these angiogenic factors preceded the involution of blood vessels. These studies indicate that therapy with anti-EGFR MAb C225 has a significant antitumor effect mediated, in part, by inhibition of angiogenesis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/terapia , Receptores ErbB/imunologia , Neovascularização Patológica , Animais , Anticorpos Monoclonais Humanizados , Carcinoma de Células de Transição/irrigação sanguínea , Carcinoma de Células de Transição/metabolismo , Divisão Celular/efeitos dos fármacos , Cetuximab , Regulação para Baixo , Fatores de Crescimento Endotelial/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Interleucina-8/metabolismo , Linfocinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microcirculação/efeitos dos fármacos , Transplante de Neoplasias , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/prevenção & controle , Neoplasias da Bexiga Urinária/secundário , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.