ABSTRACT
Cyclin D1, as a regulatory factor in cell cycle, is highly expressed in many tumors, such as lung cancer, breast cancer and thyroid cancer. The aim of the present study was to study the role of Cyclin D1 in invasion and metastasis of lung cancer cells. Lung adenocarcinoma cell line A549 and squamous cell line SK-MES-1 were selected as the objects, because A549 expresses Cyclin D1 highly, and SK-MES-1 expresses lowly. Nude mice were injected with A549 or SK-MES-1 via tail vein, and were sacrificed after 4 weeks for cancer tissue isolation. The harvested cancer cells were reinjected into another nude mouse. After one more time of such seeding, highly metastatic lung cancer model was established. After A549 and SK-MES-1 were transfected with Cyclin D1 RNAi and expression vector respectively, transwell migration assay was used to analyze transferring capacity of lung cancer cells. Western blot was used to detect Cyclin D1 and WNT/TCF pathway proteins expressions in parental cell lines and cancer tissue from metastasis model animals. The results showed that, along with the increase of seeding times, lung cancer cells from model animals, no matter A549 or SK-MES-1, exhibited augmented metastasis activity and up-regulated Cyclin D1 expression. The transferring capacity was weakened significantly in A549 cells where the Cyclin D1 was interfered by RNAi, and it was enhanced significantly in SK-MES-1 cells which were transfected with the expression vector of Cyclin D1. The expressions of WNT/TCF pathway proteins, including β-catenin, lymphoid enhancer-binding factor (LEF) and T cell factor (TCF), increased significantly in highly metastatic model animals. The parental cell lines showed lower expressions of WNT/TCF pathway proteins compared with cancer tissue from metastasis model animals. These results suggest that Cyclin D1 is closely related with the invasion and metastasis of lung cancer cells, and the WNT/TCF signal pathway may promote the expression of Cyclin D1.
Subject(s)
Animals , Female , Humans , Male , Mice , Adenocarcinoma , Metabolism , Pathology , Carcinoma, Squamous Cell , Metabolism , Pathology , Cell Line, Tumor , Cyclin D1 , Genetics , Metabolism , Lung Neoplasms , Metabolism , Pathology , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , RNA Interference , Transfection , Wnt Signaling PathwayABSTRACT
To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.
Subject(s)
Humans , Adenocarcinoma , Pathology , Apoptosis , Cell Line, Tumor , Cyclin D1 , Genetics , DNA, Antisense , Pharmacology , Genetic Vectors , Lung Neoplasms , Pathology , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Recombination, Genetic , Retinoblastoma Protein , Metabolism , Transfection , Vascular Endothelial Growth Factor A , MetabolismABSTRACT
PURPOSE: The purpose of this study is to explore the dynamic change of brain-derived neurotrophic factor (BDNF) mRNA, protein, and tyrosine kinase-coupled receptor (TrkB) mRNA of the rat hippocampus under different stress conditions and to explore the influence of senescence on the productions expression. MATERIALS AND METHODS: By using forced-swimming in 4degrees C cold ice water and 25degrees C warm water, young and aged male rats were randomly divided into acute stress (AS) and chronic mild repeated stress (CMRS) subgroups, respectively. BDNF productions and TrkB mRNA in the hippocampus were detected by using Western-blotting and reverse transcription-polymerase chain reaction (RT-PCR), separately, at 15, 30, 60, 180, and 720 min after the last stress session. RESULTS: The short AS induced a significant increase in BDNF mRNA and protein in both age groups, but the changes in the young group were substantially greater than those of the aged group (p < 0.005). The CMRS resulted in a decrease in BDNF mRNA and protein, but a significant increase in TrkB mRNA in both young and age groups. The expression of BDNF mRNA and protein in the AS groups were higher than in the CMRS groups at 15, 30, and 60 min after stress. CONCLUSION: The results indicated that the up/down-regulation of BDNF and TrkB were affected by aging and the stimulus paradigm, which might reflect important mechanisms by which the hippocampus copes with stressful stimuli.