RESUMO
In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the "ladder pattern" revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Carcinoma/patologia , Heparina/farmacologia , Neoplasias Nasofaríngeas/patologia , Carcinoma/metabolismo , Carcinoma/ultraestrutura , Humanos , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/ultraestrutura , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
OBJECTIVE: To compare the variation in morphology of low and high metastatic variants C6 and C7, which were isolated from mouse lung adenocarcinoma cell line (LA795) by single cell culture technique. METHOD: The low and high metastatic variants C6 and C7 were used in vitro, to compare the variation in morphology, cytoskeleton, motility, adhesion under the action of LN molecule and to compare the variation in the expression of endogenous LN molecule as well. RESULTS: Under LN molecule free culture condition, the motility and adhesion of high metastatic cell C6 were lower than those of low metastatic cells C7. This phenomenon indicated that the metastatic potential in vivo might be of negative correlation to the motility or adhesion in vitro. But if the LN molecules were added to the culture for 48 hours. Significantly, the motility and the adhesion were enhanced, the cytoskeleton network was reorganized, and the morphology was changed. In the C6 and C7 cells, both the motility and adhesion in vitro were positively correlated with the metastatic potential in vivo under the action of LN molecule. In addition, the distribution of the cytoskeleton network in the high metastatic variant C6 was uneven, irregular, close to one side of cell nucleus. The high metastatic cells C6 were shown strongly positive for endogenous LN by immunohistochemical technique and the distribution of the endogenous LN was close to one side of the cell nucleus. CONCLUSIONS: The quantity and distribution of endogenous and exogenous LN molecule and its receptors on the tumor cells may be an important factor for tumor metastatic heterogeneity. The mechanism of the action of LN molecule is correlated with the reorganization of the cytoskeleton.
Assuntos
Adenocarcinoma/patologia , Movimento Celular/efeitos dos fármacos , Laminina/farmacologia , Neoplasias Pulmonares/patologia , Animais , Adesão Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Laminina/fisiologia , Camundongos , Metástase Neoplásica , Receptores de Laminina/metabolismo , Células Tumorais CultivadasRESUMO
OBJECTIVE: To study methods for blocking tumor metastasis. METHODS: Endogenous laminin (LN), protein kinase C (PKC) inhibitor staurosporine (SP), anti-LN receptor (LN-R) were used to act on murine fibrosarcoma cells which were strongly positive for endogenous LN and LN-R, then infused into the caudal veins of 77 C57 mice once, to initiate lung tumor formation. Before infusion, the cells were all measured for surface LN-R quantity with flow cytometry. RESULTS: The positive rate of LN-R on tumor cell surface in the control group, LN group, SP group, anti-LN-R group and anti-LN group being 56.5%, 76.3%, 50.2%, 31.9% and 20.6% respectively. The average volume of the tumors in the lungs of mice being 76 mm3, 110 mm3, 14 mm3, 5 mm3 and 20 mm3 respectively. The rate of tumor embolus formation in lymphatic and blood vessels being 81%, 94%, 36%, 43% and 68% respectively. CONCLUSION: The pathological changes and metastasis in the LN group are more svere than that in control group. But all other groups are less severe than the control group. The level of LN-R on tumor cell surface is positively correlated with tumor volume and rat of tumor embolus formation. The lipositol conduction system in tumor cells is also closely related with size of tumor foci and rate of tumor embolus formation.
Assuntos
Fibrossarcoma/metabolismo , Laminina/farmacologia , Receptores de Laminina/metabolismo , Estaurosporina/farmacologia , Animais , Feminino , Fibrossarcoma/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Proteína Quinase C/antagonistas & inibidores , Distribuição Aleatória , Células Tumorais CultivadasRESUMO
Anti-bFGF serum was used to treat rabbit experimental silicosis. At 90 days, when experimental group was compared with control group, the number and size of the nodules in experimental group were much less and smaller than those in control group. Nodules in control group were mainly consist of fibroblast mixed with a small amount of collagen fiber, whereas nodules in experimental group were made up of macrophages. At 180 days, in addition to fibroblasts, there was II to III degree of collagen fiber in nodules of control group. Most of nodules in experimental group mainly contained macrophages. Fibroblasts of lung in fetal mice were cultured in vitro. The results showed that bFGF stimulated proliferation of fibroblast, whereas anti-bFGF serum inhibited fibroblast growth. It appeared that anti-bFGF serum actually blocked proliferation of fibroblast and fibrosis in rabbit experimental silicosis.
Assuntos
Fator 2 de Crescimento de Fibroblastos/imunologia , Silicose/terapia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Soros Imunes/farmacologia , Imunização Passiva , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Silicose/patologiaRESUMO
The current study shows that a clonal derivative of the Jurkat cell line up-regulates both the avidity and density of the alpha 6/beta 1 receptor in response to phorbol 12-myristate 13-acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up-regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexamide, can be blocked by monoclonal antibodies (Mabs) to the beta 1 and alpha 5 subunits of the beta 1 family of integrins, and is not associated with increased expression of the alpha 5 or beta 1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48-72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the beta 1 and alpha 6 subunits, and is associated with increased expression of the alpha 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB-2, CEM, Molt-4, and HPB-ALL T-lymphoblastic cell lines also up-regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up-regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4-120 hours of treatment. The most mature lines (Jurkat and HPB-ALL) up-regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt-4, and HSB-2). In summary, clonal derivatives of the Jurkat cell line up-regulated attachment to laminin through protein kinase dependent increases in alpha 6/beta 1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T-lymphoblastic cell lines.
Assuntos
Fibronectinas/metabolismo , Laminina/metabolismo , Receptores de Fibronectina/metabolismo , Receptores de Laminina/metabolismo , Linfócitos T/metabolismo , Alcaloides/farmacologia , Adesão Celular , Humanos , Estaurosporina , Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Several variant cell lines that are deficient in surface laminin have been isolated from heterogeneous murine tumour populations. The parent populations from which these variant lines were isolated contain cells that express high levels of surface laminin as indicated by immunofluorescence, immunoperoxidase/electron microscopy and immunoprecipitation. The laminin-deficient lines were compared with their respective parent populations for motility by both the Boyden Chamber assay and the agarose assay. In both assays, the laminin-positive populations were much more motile than the laminin-deficient lines. The addition of exogenous laminin to the laminin-deficient lines significantly increased their motility. These observations are of interest since cell motility is thought to contribute to tumour cell metastasis, and the laminin-positive cell populations are highly tumorigenic and metastatic, while the laminin-deficient cells are of low tumorigenicity and are virtually non-metastatic.
Assuntos
Fibrossarcoma/patologia , Laminina/farmacologia , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Métodos , Camundongos , Microscopia de Contraste de Fase , Sefarose , Estimulação QuímicaRESUMO
Human epidermal melanocytes were treated with all-trans-retinoic acid (RA) and examined for adhesion to bovine serum albumin-, fibronectin- and laminin-coated culture dishes. Control and treated cells were also examined for motility into micropore filters coated with the same proteins. Treatment of the cells with 3 x 10(-6) M RA for 3-4 days resulted in inhibition of attachment to all three substrates. Decreased attachment was observed within 1.5 h. Inhibition of attachment was not due to toxicity because differences between control and treated cells disappeared by 18 h, when most of the cells (approximately 75%) were attached and spread on all three substrates. The same treatment that inhibited adhesion also reduced migration into the interstices of micropore filters coated with the same three proteins. In additional experiments, human and mouse melanoma cell lines were examined in place of normal melanocytes. RA treatment also blocked adhesion and motility of these cells. The malignant melanoma cells were less sensitive to RA than normal melanocytes in the adhesion assay but were equally sensitive in the motility assay. The ability of RA to inhibit melanocyte adhesion and motility as well as melanocyte growth could explain, in part, the capacity of retinoids to modulate melanocyte function in hyperpigmented skin lesions.