ABSTRACT
<p><b>BACKGROUND</b>nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.</p><p><b>METHODS</b>The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods.</p><p><b>RESULTS</b>The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01).</p><p><b>CONCLUSIONS</b>Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.</p>
ABSTRACT
nm23-H1 is a proven tumor metastasis suppressive gene, tumor metastasis phenotype could be reversed by transfected nm23-H1 cDNA. This study was conducted to transfect nm23-H1 cDNA into L9981 cells and to explore the function of nm23-H1 in reversing the malignant phenotype of L9981 cells. The plasmid of pLXSN-nm23-H1-EGFP was constructed by gene clone technique, and the transfected nm23-H1 cDNA cell lines of L9981-nm23-H1 was established. The protein expression of nm23-H1 was detected by Western blot. The biologic features of L9981-nm23-H1 cells were studied in vitro and in vivo. The results showed that the fusion protein of nm23-H1-EGFP was stable, continuous and expressed with high efficiency in L9981-nm23-H1 cells. The cell proliferation, colon formation and invasive ability are significantly lowered in L9981 cells transfected nm23-H1 cDNA (P < 0.01); the tumorgenesis and the lung metastasis incidence was lower in tranfected nm23-H1 cells than in L9981 and L9981-Plxsn in nude mice (P < 0.01); the rate for inhibiting tumorgenesis of nm23 -H1 was 82.56%. These data suggest that the malignant phenotype could be reversed by wild nm23-H1 gene in L9981 cells.
Subject(s)
Animals , Humans , Mice , Cell Line, Tumor , Cell Proliferation , Lung Neoplasms , Pathology , Mice, Nude , NM23 Nucleoside Diphosphate Kinases , Genetics , Neoplasm Metastasis , Neoplasm Transplantation , Recombinant Fusion Proteins , Genetics , Transfection , Tumor Suppressor Proteins , GeneticsABSTRACT
<p><b>BACKGROUND</b>It has been known that oncogenesis and development of lung can- cer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may play a very important role in these procedures. The aim of this study is to investigate the influence of exogenous MEK1/2 pathway inhibitor U0126 on the expression and activity of extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.</p><p><b>METHODS</b>The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 (parent cell line with nm23-H1 gene hetero-deletion ) were detected by Western blot and immuno-precipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abilities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods.</p><p><b>RESULTS</b>The ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentration groups of U0126 (P < 0.01), but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 (P=0.387). After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups (P < 0.01). But no significant difference of total ERK1/2 expression level was found among different time groups (P=0.689). The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose- and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration (P < 0.001). The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concentration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20μmol/l of U0126 (P > 0.05). A highly significant difference was observed when U0126 concentration increased to 40 and 60μmol/l compared with 0, 10 and 20μmol/l of U0126 (P < 0.001).</p><p><b>CONCLUSIONS</b>The inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high-metastatic large cell lung cancer cell line L9981 is dose-dependent and time-dependent. Suppressing or blocking of Ras-to-MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.</p>
ABSTRACT
<p><b>BACKGROUND</b>It has been proved that tumor development and metastasis are dependent on angiogenesis. Suppression of tumor angiogenesis can inhibit tumor growth and metastasis. Collagen X VIII/endostatin is one of the most effective inhibitors of angiogenesis at present. The aim of this study is to study the relationship between transcription expression of endostatin mRNA and clinical and pathophysiological characteristics in non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The transcription expression of endostatin mRNA was detected in 46 lung cancer tissues and paracancerous lung tissues, 14 benign pulmonary lesion tissues as control by RT-PCR method.</p><p><b>RESULTS</b>(1)The transcription expression of endostatin mRNA in lung cancer tissues (0.872±0.071) was significantly higher than that in paracancerous tissues (0.717±0.073) and benign pulmonary lesion tissues (0.611±0.026) (P < 0.001).(2)The transcription expression of endostatin mRNA in lung cancer tissues was closely related to P-TNM stages, distant metastasis, grade of cell differentiation and size of the primary tumors (P < 0.05), but not to location of tumor, lymph node status, histological classification, age and sex of the patients and smoking or not (P > 0.05).</p><p><b>CONCLUSIONS</b>The transcription expression of endostatin mRNA in NSCLC tissues is significantly higher than that in paracancerous tissues and benign pulmonary lesion tissues, and is closely related to P-TNM stages, distant metastasis, grade of cell differentiation and size of the primary tumors, hence it might be helpful to evaluate the biological behavior of lung cancer.</p>
ABSTRACT
<p><b>BACKGROUND</b>To investigate the influence of the tumor metastasis suppressor gene nm23 H1 on the activity of extracellular signal-regulated protein kinase (ERK) in human high metastasis large cell lung cancer cell line L9981.</p><p><b>METHODS</b>The levels of total ERK1/2 and phospho-pERK1/2 were determined with p44/42 MAP kinase antibody and dually phosphospecific phospho-44/42 MAP kinase antibody in human high-metastasis large cell lung cancer cell lines L9981 (cell line with nm23-H1 gene deletion), L9981-nm23-H1 (cell line with nm23-H1 transfected ) and L9981-PLXSN (cell line with vector transfected) by Western blot method, respectively. The activity of phospho-ERK1/2 was determined with an ERK1/2 assay kit by immunopreciptation and Western blot analysis.</p><p><b>RESULTS</b>The expression levels of phospho-ERK1/2 kinase and the activity of phospho-ERK1/2 in the lung cancer cell line L9981-nm23-H1 were remarkably higher than those of the L9981 cell line and L9981-PLXSN cell line ( P < 0.01), but no significant difference in both the phospho-ERK1/2 expression and phospho-ERK1/2 activity was observed between the L9981 and L9981-PLXSN cell lines ( P > 0.05). There was no significant difference in the total ERK1/2 level among the three cell lines.</p><p><b>CONCLUSIONS</b>nm23-H1 gene can obviously targetly suppress the activity of ERK1/2 in human high metastasis large cell lung cancer cell line L9981. This suggest that the mechanisms of nm23-H1 gene as a tumor metastasis suppressor gene may be related to its suppression to the MAPK/ERK signal transduction pathway.</p>
ABSTRACT
<p><b>BACKGROUND</b>To study the regulation of nm23-H1 gene on the expression of integrins in human high-metastasis large cell lung cancer cell line L9981 by transfecting nm23-H1 into cell.</p><p><b>METHODS</b>Lipofect was used to transfect nm23-H1 into the L9981 cell line; semi-RT-PCR was used to detect the difference of integrin β1 and integrin β3 mRNA expressions between tranfected and non-transfected cell lines; flow cytometry was used to detect the difference of integrin β1 and integrin β3 protein expressions between transfected and non-transfected cell lines.</p><p><b>RESULTS</b>(1) A transgenic cell line L9981-nm23-H1 was obtained, which could stably and effectively express nm23-H1. The mRNA expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were remarkably lower than those in L9981 cell line. (2) The protein expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were significantly lower than those in L9981 cell line too (P < 0.01).</p><p><b>CONCLUSIONS</b>Transfection of nm23-H1 gene into the L9981 cell line can significantly down-regulate the mRNA and protein expressions of integrin β1 and β3 in this cell line, which indicates that nm23-H1 may reverse the metastasis potential of L9981 cell line through modulation of integrin expression.</p>
ABSTRACT
<p><b>BACKGROUND</b>To construct recombinant adenoviral vector carrying Smad3D or Smad7 by a simplified means.</p><p><b>METHODS</b>Based on AdEasy System, adenoviral backbone plasmid vector and shuttle vector carrying the gene of interest were transferred into E.coli BJ5183 by chemical transformation methods in special order. The homologous recombination was performed.</p><p><b>RESULTS</b>Recombinant adenoviral vector pAd-Smad3D and pAd-Smad7 were constructed successfully, which were confirmed by restriction enzyme digesting.</p><p><b>CONCLUSIONS</b>Recombinant adenoviral vector may be constructed quickly and efficiently in E.coli by sequential chemical transformation methods.</p>
ABSTRACT
<p><b>BACKGROUND</b>To construct the recombinant adenovirus of mutant k-ras by using the method of homogenous recombination in bacteria.</p><p><b>METHODS</b>Mutant k-ras gene was liberated from the vector of pcDNA3-k-ras 12(Val) via KpnI+XhoI digestion, and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack CMV/k-ras 12(Val). Then it was linearized with PmeI and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with PacI and transfected to 293 cells to package adenovirus. The PCR technique was used to detect target gene. The titre and its infection rate of the Ad-k-ras 12(Val) was measured with the aid of GFP expression.</p><p><b>RESULTS</b>There were over 25% positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack-CMV/k-ras 12 (Val) and pAdEasy-1 by method of CaCl₂. PCR test indicated each the recombinant adenovirus contained the insert of k-ras 12(Val). The titre of purified recombinant adenovirus was 1.2×10¹² pfu/ml.</p><p><b>CONCLUSIONS</b>The method of homologous recombination in bacteria is convenient and efficient, which compared with that of in cell and the pepared Ad-k-ras 12(Val) paves a sound foundation for further study.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To explore the role of fragile histidine triad(FHIT) gene in the proliferation, apoptosis and tumorigenesis of human lung cancer cells.</p><p><b>METHODS</b>FHIT gene packaged with lipofectin was transfected into the cells of a human lung adenocarcinoma cell line (A549), which stably expressed ectogenous FHIT gene. The FHIT mRNA and protein expression of A549-FHIT, A549-vector and A549 cell were detected by reverse transcription-PCR(RT-PCR), Western blot and immunocytochemical methods. The cell cycle pattern and apoptosis were assayed by using flow cytometry.</p><p><b>RESULTS</b>After transfection of FHIT gene, cell growth was obviously inhibited (P<0.01). The apoptosis index of A549-FHIT (8.42%) was significantly higher than that of A549-vector (5.45%) and A549 cells (5.71%)(P<0.01). The clone-formation rate of A549-FHIT cell (21.84%) was significantly lower than that of A549-vector (28.70%) and A549 cells (31.68%, P<0.01). Compared with control cell lines, larger scale of A549-FHIT cells accumulated in G0/G1, presenting that the proportion of the cells in G0/G1 phase was obviously increased from 67.78 % to 82.35 %. Tumorigenicity of the A549 cells in nude mice was greatly inhibited by expression of ectogenous FHIT gene, the weight and volume of A549-FHIT(1.61 g/1.37 cm(3)) were significantly lower than that of A549-vector (2.45 g/1.99cm(3)) and A549 cells (2.77 g/2.27 cm(3))(P<0.05).</p><p><b>CONCLUSION</b>Expression of ectogenous FHIT gene can obviously inhibit the proliferation and tumorigenesis of A549 cells, and can induce A549 cells into programmed cell death. The result of this study suggests that FHIT gene may be a tumor suppressor gene in human lung cancer cells.</p>