RESUMEN
It is extensively shown that integrin can regulate various cellular functions, including apoptosis, probably by contributing to signal transduction processes through interaction with extracellular matrix (ECM) proteins. In the present study, DNA flow cytometric analysis demonstrated that SMMC-7721 hepatocarcinoma cells treated with 80 microM all-trans-retinoic acid (atRA) showed an increased expression of the integrin alpha5beta1, which was associated with the growth inhibition of the cells. We found that atRA treated cells showed obvious apoptosis. Then, it was postulated that the enhanced content of integrin alpha5beta1 in the absence of ligation with fibronectin (Fn) would stop transducing survival signals, and lead to decreased cell growth and apoptosis. To elucidate this hypothesis, we cultured the atRA treated cell in L-poly-lysine-coated and Fn-coated flask, respectively. The results indicated that Fn binding prevented the cells from apoptosis induced by atRA, in contrast to L-poly-lysine binding. When the transfectant with enhanced expression of integrin alpha5beta1 at the same level of atRA treated cell was cultured in L-poly-lysine-coated flask, apoptosis was triggered. However, apoptotic cell was not detected when those cells were cultured in Fn-coated flask. Meanwhile, culturing the transfectant in the antibody (against integrin alpha5 subunit)-coated flask induced 18.33% of the cells into apoptosis, which is far more than the control group. Our study suggests that increased expression of integrin alpha5beta1 on the surface of SMMC 7721 hepatocarcinoma cell treated by atRA, when unbound to Fn, would stop transducing survival signals to lead to "anoikis", and can be reverted by the interaction of integrin alpha5beta1 with Fn.
Asunto(s)
Anoicis/efectos de los fármacos , Receptores de Fibronectina/genética , Tretinoina/farmacología , Antígenos CD/genética , Carcinoma Hepatocelular , División Celular/fisiología , Fibronectinas/fisiología , Humanos , Etiquetado Corte-Fin in Situ , Integrina alfa5 , Integrina beta1/genética , Cinética , Neoplasias Hepáticas , Subunidades de Proteína , Proteínas Recombinantes/biosíntesis , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
The purpose of this paper is to study the effect of N-acetylglucosaminyltransferase V (GnT-V) overexpression on the migration of 7721 cells and its mechanism. The abilities of migration of both 7721 cells transfected with GnT-V cDNA and 7721 cells transfected with pcDNA3 was detected, the expressions of integrin and E-cadherin which are important adhesion molecules on surface membrane and closely related to the abilities of invasion and metastasis. Cell migration abilities were measured by the agarose drop explant method. Flow cytometric analysis (FACS) was applied to determine the relative amounts of integrin alpha 5 and beta 1 subunits on the cell surface while RTPCR was carried out to determine the expression of their mRNA. The expression of E-cadherin was examined by the immunocytochemical ABC method. Western blot analysis was carried out to examine the expression of beta-catenin. GnT-V overexpression enhanced evidently the migration ability of 7721 cells and increased the amount of integrin alpha 5 subunit to 2.9 times of that of control while the amount of beta 1 subunits was not significantly changed. Besides, the expressions of E-cadherin and beta-catenin were enhanced at different levels in GnT-V/7721 cells compared with mocked. The results suggested that the overexpression of GnT-V related to the production of N-linked sugar chains could promote the expressions of integrin, E-cadherin and beta-catenin on 7721 cells so that the migration ability of tumor cells was enhanced.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Neoplasias Hepáticas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Cadherinas/biosíntesis , Proteínas del Citoesqueleto/biosíntesis , Humanos , Integrina alfa5beta1/biosíntesis , N-Acetilglucosaminiltransferasas/biosíntesis , ARN Mensajero/biosíntesis , Transactivadores/biosíntesis , Células Tumorales Cultivadas , beta CateninaRESUMEN
Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Fibronectina/biosíntesis , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor , Carcinoma Hepatocelular/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica/efectos de los fármacos , Humanos , Laminina/metabolismo , Neoplasias Hepáticas/patología , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/biosíntesis , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores de Fibronectina/genética , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Integrin alpha 5 beta 1 and alpha 2 beta 1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin alpha 5 beta 1 was decreased and another integrin alpha 6 beta 1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singly transfected with integrin alpha 5 and/or beta 1 cDNAs were established, and designated alpha 5 beta 1.6-7721, alpha 5.3-7721, and beta 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin alpha 5 and beta 1 subunits resulted in the over expression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in alpha 5.3-7721, beta 1.6-7721, alpha 5 beta 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with beta 1.6-7721, and alpha 5 beta 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin alpha 5 beta 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin alpha 5 beta 1 in malignant cells could be a potential means of treating hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Receptores de Fibronectina/genética , Animales , Pruebas de Carcinogenicidad , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ciclo Celular , División Celular , Ensayo de Unidades Formadoras de Colonias/métodos , Citometría de Flujo , Humanos , Inyecciones Subcutáneas , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Receptores de Fibronectina/biosíntesis , TransfecciónRESUMEN
PTEN/MMAC1/TEP1 is a tumor suppressor gene. Its mutation has been found in several different types of human cancers. 34 primary human hepatocellular carcinomas have been examined for mutations in exon 5 and exon 8 of the PTEN gene. Exon 5 and exon 8 were amplified by polymerase chain reaction (PCR) with intronic primers and subjected to single strand conformation polymorphism (SSCP) analysis. SSCPs were found in 4 of the 34 hepatocellular carcinomas analyzed. Direct sequencing of the PCR products identified single base-pair substitutions in the four tumor DNA samples, two in intron 4 and two in exon 8. One of the base-pair substitution in exon 8 is a missense mutation, which changed codon 304 of PTEN protein from Cys to Gly. These data suggest that PTEN may be involved in the carcinogenesis and development of hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/genética , Genes Supresores de Tumor , Neoplasias Hepáticas/genética , Mutación , Exones/genética , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
In order to investigate whether TGF-beta 1 could rapidly regulate integrin induced signaling, we treated SMMC-7721 human hepatocellular carcinoma cells with human recombinant TGF-beta 1 for 10 min, and examined cell adhesion, integrin amount and FAK tyrosine phosphorylation. We used cell adhesion assay to estimate the affinity of alpha 5 beta 1 integrin with fibronectin, and analyzed the amount of integrin alpha 5 and beta 1 subunits by performing FACS analysis. Then western blot analysis was carried out to examine tyrosine phosphorylation level of FAK. Our results showed that TGF-beta 1 could rapidly attenuated cell adhesion onto Fn without changing the expression of alpha 5 beta 1 integrin, and at the meantime dephosphorylated FAK. It suggested that TGF-beta 1 rapidly regulated the activation of integrin, and stimulated FAK dephosphorylation, which might induce depolarization in SMMC-7721 hepatocellular carcinoma cells, then facilitates the detachment of tumor cells at early stages of migration.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Fibronectinas/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacología , Carcinoma Hepatocelular/patología , Adhesión Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrina alfa5beta1/biosíntesis , Neoplasias Hepáticas/patología , Fosforilación/efectos de los fármacos , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
The gene expression of integrin a5 beta 1 and the ability of cell adhesion of SMMC-7721 hepatocellular carcinoma cell are reported to be altered in the presence of 100nM phorbol 12-myristate-13-acetate (PMA). The ability of cell adhesion to extracellular matrix-fibronectin (Fn) was 18.8%, 38.7% and 56.6% enhancement for 30.60 and 120 mins treatment with 100nM PMA, respectively, as compared with the PMA control. But after 6 and 12 hours, it decreased to 57.4% and 61.1%, whereas the adhesion to polylysine remained the same. Using sufficient amount of anti-a5 and anti-beta 1 mAb to pre-block the adhesion sites we found that cell adhesion to Fn was inhibited by approximately 40%. It seems that other integrins mediate cell adhesion to Fn besides alpha 5 beta 1. The results obtained from the Northern blot with alpha 5 cDNA probe showed that PMA could downregulate the transcription of integrin alpha 5 beta 1 gene from 10 mins to 12 hours. The transcription was lowered to 16.9% of the control after 30 mins. The inhibition rate was still 43.6% after 12-hour treatment. The possible mechanism by which PMA influences the cell adhesion and integrin gene expression is discussed here.
Asunto(s)
Carcinógenos/farmacología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Acetato de Tetradecanoilforbol/farmacología , Northern Blotting , Carcinoma Hepatocelular/metabolismo , Adhesión Celular/efectos de los fármacos , Fibronectinas/metabolismo , Humanos , Integrinas/biosíntesis , Neoplasias Hepáticas/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Tumor invasion and metastasis are complex processes, requiring the ability of tumor cells to interact with proteins of the extracellular matrix through cell-adhesion molecules on the cell surface. Integrins are heterodimeric membrane glycoproteins, consisting of alpha and beta subunits, which enable cells to recognize adhesive substrates in the extracellular matrix. The roles of the integrin alpha5beta1 in tumor invasion are highlighted by finding that some tumor cells have lost or reduced alpha5beta1 expression. It therefore functions as a negative signaling regulator. Expression of alpha5beta1 and its mediation of cell adhesion in hepatocellular carcinoma (HCC) have not been elucidated. In surgical specimens of HCC we found, by immunohistochemistry and Northern blot analysis, that the alpha5-positive rates in cancerous tissues were lower than the corresponding rates in non-cancerous tissues. Reduced expression of the integrin alpha5 occurred more frequently in HCC with more malignant phenotypes, such as poor differentiation, large size (more than 10-cm in diameter), absence of capsule and high invasion. Reverse transcription/polymerase chain reaction, a more sensitive assay, was used to detect the alpha5 mRNA level in LCID20, a highly metastatic model of human HCC, and LCID35, a low-metastasis model. The results showed that integrin alpha5 was negative in the former and positive in the latter. Cell adhesion assays showed the maximal percentage inhibition of anti-alpha5 mAb on SMMC 7721 cell adhesion to fibronectin to be 68.9 +/- 4.9% at the saturation concentrations of each antibody (200 microg/ml). If anti-alpha5 mAb was combined with anti-beta1 mAb, the inhibition was 74.1 +/- 11.1%. It is concluded that reduced expression of the integrin alpha5 subunit is correlated with more malignant phenotypes of human HCC. Any change in the adhesion of hepatocellular carcinoma cells to fibronectin is mainly dependent upon the function of the integrin alpha5beta1.