RESUMO
The purpose of this study was to determine the correlation between over-expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio-sensitivity of non-small cell lung carcinoma (NSCLC) cells. 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V-Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X-ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF-κB. Finally, to examine the effect of shNRP1 on proliferation and radio-sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1-A549) showed a significant reduction in colony-forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA-mediated NRP1 inhibition also significantly enhanced the radio-sensitivity of NSCLC cells both in vitro and in vivo. The over-expression of NRP1 was correlated with growth, survival and radio-resistance of NSCLC cells via the VEGF-PI3K- NF-κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio-sensitization of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neuropilina-1/metabolismo , Tolerância a Radiação , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Linhagem Celular Tumoral , Movimento Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação/efeitos da radiação , Indução de Remissão , Transfecção , Raios XRESUMO
OBJECTIVE: The purpose of the present study was to observe the changes in CD4+CD25+Nrp1+Treg cells after irradiation with different doses and explore the possible molecular mechanisms involved. METHODS: ICR mice and mouse lymphoma cell line (EL-4 cells) was used. The expressions of CD4, CD25, Nrp1, calcineurin and PKC-α were detected by flow cytometry. The expressions of TGF-ß1, IL-10, PKA and cAMP were estimated with ELISA. RESULTS: At 12 h after irradiation, the expression of Nrp1 increased significantly in 4.0 Gy group, compared with sham-irradiation group (P<0.05) in the spleen and thymus, respectively, when ICR mice received whole-body irradiation (WBI). Meanwhile the synthesis of Interleukin 10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) increased significantly after high dose irradiation (HDR) (> or = 1.0 Gy). In addition, the expression of cAMP and PKA protein increased, while PKC-α, calcineurin decreased at 12h in thymus cells after 4.0 Gy X-irradiation. While TGF-ß1 was clearly inhibited when the PLC-PIP2 signal pathway was stimulated or the cAMP-PKA signal pathway was blocked after 4.0 Gy X-irradiation, this did not limit the up-regulation of CD4+CD25+Nrp1+Treg cells after ionizing radiation. CONCLUSION: These results indicated that HDR might induce CD4+CD25+Nrp1+Treg cells production and stimulate TGF-ß1 secretion by regulating signal molecules in mice.
Assuntos
AMP Cíclico/metabolismo , Terapia de Imunossupressão , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas Quinases/metabolismo , Irradiação Corporal Total/efeitos adversos , Animais , Calcineurina/genética , Calcineurina/metabolismo , Relação Dose-Resposta à Radiação , Feminino , Regulação da Expressão Gênica/efeitos da radiação , Interleucina-10/genética , Interleucina-10/metabolismo , Subpopulações de Linfócitos/fisiologia , Masculino , Camundongos , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fosfoinositídeo Fosfolipase C/genética , Proteínas Quinases/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The cytokinesis-block micronucleus assay has proven to be a reliable technique for biological dosimetry. This study aimed to establish the dose-response curve for X-ray-induced micronucleus. Peripheral blood samples from three healthy donors were irradiated with various doses and scoring criteria by the micronuclei (MN) in binucleated cells. The results showed that the frequency of MN increased with the elevation of radiation dose. CABAS and Dose Estimate software were used to fit the MN and dose into a linear quadratic model, and the results were compared. The linear and quadratic coefficients obtained by the two software were basically the same and were comparable with published curves of similar radiation quality and dose rates by other studies. The dose-response curve established in this study can be used as an alternative method for in vitro dose reconstruction and provides a reliable tool for biological dosimetry in accidental or occupational radiation exposures.