RESUMO
In prostate cancer progression, the basal lamina switches from predominantly laminin-5 to laminin-10. DU-145 prostate cancer cells were treated with either soluble laminin-5 (20 ng/ml) or laminin-10 (1 microg/ml) for 6, 24, and 48 hr. Total RNA was harvested for a 7,500 human cDNA microarray. Hybridizations were carried out in accordance with a 10 sample analysis of variance (ANOVA) statistical model. One thousand one hundred sixteen genes had measurable expression 2 standard deviations above background and 50% of spots for any given sample for all hybridizations were positive. Expression values of significantly varying genes were clustered and a list of 408 genes (P < 0.05) with a 1.5 or greater fold change in at least one time point were chosen for further analysis. Seventy eight changed in a time-dependent manner with laminin-10 treatment, 85 changed with laminin-5, and 13 showed changes with both treatments. The 408 genes that passed a paired t-test in at least one time-dependent category were further analyzed using Pathway Miner. One of the largest gene association networks involved signal transduction in the growth factor-MAP kinase pathways. EGFR was validated by real-time PCR and laminin-10 mediated cell adhesion activated EGFR in DU-145 cells. Both laminins appear to be important signal transducers in prostate cancer.
Assuntos
Moléculas de Adesão Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Laminina/fisiologia , Neoplasias da Próstata/genética , Calpaína/genética , Calpaína/metabolismo , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Neoplásico/genética , Transdução de Sinais/genética , Fatores de Tempo , CalininaRESUMO
Thrombin is a serine protease that promotes platelet aggregation, blood coagulation, and tissue repair. A peptide derived from a non-proteolytically active region of thrombin, TP508, also promotes tissue repair and increased vascularity, yet does not activate platelet and inflammatory cascades. TP508 binds to cells with high affinity and stimulates cells independent of the proteolytically active thrombin receptors (PARs) and thus is considered to activate a non-proteolytically active receptor (non-PAR) pathway. Using a model of angiogenic sprouting, we further defined the angiogenic potential of TP508 and investigated the role of non-proteolytic, thrombin-mediated pathways in angiogenesis. The assay involves measuring angiogenic sprouting from cultured, intact microvessel fragments. In this assay, TP508 stimulated angiogenic sprouting to an extent similar to or greater than the potent angiogenic factor, VEGF. However, TP508 had no significant effect on the number of sprouts that formed per vessel. In contrast to TP508, proteolytically active receptor agonists had no effect or inhibited angiogenic sprouting. The increased sprouting activity stimulated by TP508 was VEGF dependent but did not involve an increase in VEGF mRNA expression above baseline levels. These results suggest that TP508 acts early in angiogenesis and directly on microvascular cells to accelerate sprouting, but not to induce more sprouting, in a manner different than the intact thrombin molecule.
Assuntos
Neovascularização Fisiológica/fisiologia , Peptídeos/metabolismo , Trombina/metabolismo , Animais , Vasos Sanguíneos/anatomia & histologia , Vasos Sanguíneos/metabolismo , Gelatinases/genética , Gelatinases/metabolismo , Técnicas In Vitro , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Ligação Proteica , Ratos , Receptores de Trombina/agonistas , Receptores de Trombina/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Interactions between extracellular matrix proteins and prostate carcinoma cells change dramatically during prostate tumor progression. We have concentrated on two key modifications that occur in the hemidesmosome in prostate carcinoma: loss of laminin-5 protein expression and altered basal cell polarity of the alpha6beta4 integrin. We previously demonstrated two cell line-specific isoforms (beta3A and beta3B) of the LAMB3 message. Cells expressing only the beta3B isoform did not translate the beta3 protein and were unable to assemble the laminin-5 trimer. One such cell line, LNCaP, was selected to determine whether restoration of the laminin-5 beta3A isoform would cause expression of a functional laminin-5 beta3 chain, assembly and secretion of the laminin-5 trimer, and reversion to a non-neoplastic phenotype. Laminin-5 beta3A cDNA was cloned and stably transfected into LNCaP cells. We observed the restoration of the beta3 protein, but a laminin-5 trimer was not secreted. Moreover, increased cell migration was demonstrated, and tumorigenicity was increased in SCID mice. A microarray analysis, performed between transfected and nontransfected LNCaP cells, showed most changing genes to be associated with signal transduction. The beta3 chain of laminin-5 may thus play an important role in signal transduction, which may enhance cell motility and tumorigenesis.