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cDNA microarray technique on screening multi-drug resistance-related genes of human non-small cell lung cancer.

Meiyan LIU ; Chunhong LI ; An YAN ; Li CAI

Chinese Journal of Lung Cancer.2010;13(4):322-325. doi:10.3779/j.issn.1009-3419.2010.04.10

BACKGROUND AND OBJECTIVEThe aim of this study is to screen for multi-drug resistance-related genes of human non-small cell lung cancer (NSCLC), and provide the evidences for drug-sensitive predicting genes of different NSCLC patients treated with chemotherapeutic drugs.

METHODSSensitivity and inhibition ratio of five antitumor drugs (NVB, GEM, TAL, DOC, CDDP) on 75 fresh NSCLC samples from different individuals were studied by means of culturing primary tumor cells and MTT assay. After the five chemotherapeutic drugs were used, multi-drug resistance-related genes of NSCLC with cDNA microarry on the samples which were all high sensitive and those resistant were screened.

RESULTScDNA microarray analysis screened out 212 genes, 168 of which were up-regulated while the other 44 were down-regulated in the group of highly sensitive compared with the group of resistance.

CONCLUSIONThe multi-drug resistance of NSCLC may be correlative with the 212 genes screened by cDNA microarray; the detailed mechanisms of the genes still need to be detected in the future.

Antineoplastic Agents ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; genetics ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; Oligonucleotide Array Sequence Analysis

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Expression and clinicopathologic significance of human achaete-scute homolog 1 in pulmonary neuroendocrine tumors.

Fei LI ; Zhiyong ZHONG ; Rui LI ; Heyu HUANG ; Lijun WANG ; Donghan ZHENG ; Daorong ZHANG

Chinese Journal of Lung Cancer.2010;13(4):317-321. doi:10.3779/j.issn.1009-3419.2010.04.09

BACKGROUND AND OBJECTIVEHuman achaete-scute homolog 1 (hASH1) gene plays a critical role in development of the central nervous system, automatic nervous system, adrenal medullary chromaffin cells, thyroid C cells and pulmonary neuroendocrine cells. The aim of this study is to determine hASH1 gene expression in the normal lung tissue and various types of lung tumors, to analyze whether its expression correlated with pulmonary neuroendocrine markers, and to explore the possibility of hASH1 as clinical pathological markers in the neuroendocrine tumors compared with previous neuroendocrine tumor markers.

METHODShASH1, Chromogranin A, Synaptophysin and CD56 expression were examined in lung tumor specimens (lung inflammatory pseudotumor, squamous cell carcinoma, adenocarcinomas, large cell carcinoma, typical carcinoids, atypical carcinoids, large cell neuroendocrine carcinomas and small cell lung carcinoma and corresponding normal lung specimens) using immunohistochemistry (S-P method). Western blot and reverse transcription polymerase chain reaction (RT-PCR) assay were applied to detect the expressions of hASH1 protein and mRNA in lung cancer tissues.

RESULTShASH1 expression was positive in 2/16 (12.5%) typical carcinoids, 15/20 (75%) atypical carcinoids, 6/10 (60%) large cell neuroendocrine carcinomas and 31/40 (77.5%) small cell lung carcinoma, respectively, but not in any normal lung tissue (0/10), lung inflammatory pseudotumor (0/49), squamous cell carcinoma (0/30), adenocarcinomas (0/30) or large cell carcinoma (0/20). There was a significant difference in hASH1 expression between typical carcinoids and atypical carcinoids (P < 0.01), but not in large cell neuroendocrine carcinomas and small cell lung carcinoma (P > 0.05). hASH1 expression highly closely correlated with Chromogranin A, Synaptophysin and CD56 expression (P < 0.05).

CONCLUSIONhASH1 is a new kind of highly specific markers of pulmonary neuroendocrine tumours, and may be applied to clinical pathology diagnosis of the pulmonary neuroendocrine tumors.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Carcinoma, Large Cell ; genetics ; metabolism ; pathology ; Carcinoma, Neuroendocrine ; genetics ; metabolism ; pathology ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Humans ; Immunohistochemistry ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Neuroendocrine Tumors ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; genetics ; metabolism ; pathology

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Prognostic value of methylation status of RASSF1A gene as an independent factor of non-small cell lung cancer.

Hui ZHANG ; Shucai ZHANG ; Zongde ZHANG ; Hongyan JIA ; Shuxiang GU ; Dan ZHAO

Chinese Journal of Lung Cancer.2010;13(4):311-316. doi:10.3779/j.issn.1009-3419.2010.04.08

BACKGROUND AND OBJECTIVEPromoter hypermethylation of the RASSF1A gene is among the most abundant epigenetic deregulations in human cancer. The aim of this study is to investigate the relationship between the methylation status of RASSF1A promoter and the prognoses of non-small cell lung cancer (NSCLC).

METHODSThe methylation status of RASSF1A promoter in 150 NSCLC and 25 non-malignant tissues was determined using a methylation-specific polymerase chain reaction (MSP).

RESULTSRASSF1A promoter hypermethylation was detected in 38.7% (58/150) of NSCLC tissues, but in none of the non-malignant tissues. The patients with hypermethylation of RASSF1A had a poor survival rate, and the relationship between the survival rate and hypermethylation of RASSF1A was statistically significant (P = 0.004). Then by using stepwise Cox proportional hazard regression testing, methylation status of RASSF1A was an independent factor affecting the NSCLC patients' survival (RR = 1.584, 95% CI: 1.040-2.411, P = 0.032).

CONCLUSIONThe hypermethylation of the RASSF1A promoter may be an independent prognostic factor of NSCLC after operation.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; DNA Methylation ; genetics ; Female ; Humans ; Lung Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Prognosis ; Promoter Regions, Genetic ; genetics ; Tumor Suppressor Proteins ; genetics

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99Tc(m) -N(NOEt)2 uptake kinetics difference among KMB17 human embryonic lung diploid fibroblast and different human lung cancer cells.

Tiekun MA ; Jianming CAO ; Wei JIA

Chinese Journal of Lung Cancer.2010;13(4):307-310. doi:10.3779/j.issn.1009-3419.2010.04.07

BACKGROUND AND OBJECTIVEPET/CT imaging is expensive, so searching the tumor imaging agent for SPECT/CT is necessary. 99Tc(m) -N(NOEt)2 [bis (N-ethoxy-N-ethyl dithiocarbamato) nitrido 99Tc(m) (V)] can be uptaken by lung cancer cells and other cells alike. The aim of this study is to evaluate the distinctive value in lung tumor with 99Tc(m) -N(NOEt)2, the difference in its uptake kinetics in human embryonic lung diploid fibroblasts KMB17 and several kinds of lung cancer cells lines.

METHODSFirstly, six different cell culture medium which contained YTMLC Gejiu human lung squamous carcinoma cell, SPC-A1 human lung adenocarcinoma cell, AGZY low metastatic human lung adenocarcinoma, 973 high metastatic human lung adenocarcinoma cell, GLC-82 Gejiu human lung adenocarcinoma cell, and KMB17 human embryonic lung diploid fibroblast, respectively with equal cell density of 1 x 10(6)/mL and the same volume were prepared; secondly, the same radioactive dose of 99Tc(m) -N(NOEt)2 was added into each sample and then 300 microL mixed sample was taken out respectively and cultured in 37 degrees C culture box; Finally, 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min after cultivation, centrifuged each cultured sample and determined the intracellular radiocounts of each sample, calculated each cell sample's uptake rate of 99Tc(m) -N(NOEt)2 at different time.

RESULTSStatistical difference was found among six cell samples, and the uptake rate sequence from high to low is 973 and SPC-A1 > YTMLC > GLC-82 > AGZY > KMB17 respectively; furthermore, 30 min-45 min after culture, the uptake rate reached stability, and the 45 min uptake rate of each sample was higher than its 96.7% uptake peak.

CONCLUSIONBased on the results above mentioned, it is supposed that there are discriminative clinical value when using 99Tc(m) -N(NOEt)2 as a tumor targeting imaging agent, and 30 min or so after injection may be the best imaging time in the early imaging stage.

Cell Line ; Cell Line, Tumor ; Contrast Media ; pharmacokinetics ; Fibroblasts ; cytology ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; Organotechnetium Compounds ; pharmacokinetics ; Tomography, Emission-Computed, Single-Photon

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Curcumin promoted the apoptosis of cisplain-resistant human lung carcinoma cells A549/DDP through down-regulating miR-186*.

Ni TANG ; Jian ZHANG ; Yongping DU

Chinese Journal of Lung Cancer.2010;13(4):301-306. doi:10.3779/j.issn.1009-3419.2010.04.06

BACKGROUND AND OBJECTIVECurcumin, a natural compound, is derived from the rthizom of Curcuma longa. In vitro and in vivo preclinical studies have shown its anti-inflammatory, antioxidant, anticancer activities and so on. miR-186*, which was found by microarray technology, was highly expressed in lung carcinoma cells A549/DDP. The aim of this study is to illustrate whether Curcumin could promote the apoptosis of A549/DDP cells through regulating the expression of miR-186*.

METHODSAn oligonucleotide microarray chip was used to profile microRNA (miRNA) expressions in A549/DDP cells treated with and without Curcumin. The significantly differentially expressed miRNA, which was selected from microarray chip, validated by quantitative real-time PCR. Ultimately, the remarkably expressed miRNA modulated the apoptosis assaying by flow cytometry expriments and the survival rate was measured by MTT method.

RESULTSThe microarray chip results demonstrated: Curcumin altered the expression level of miRNAs compared with untreated control in A549/DDP cell line, miR-186* was significantly down-regulated after Curcumin treatment, which confirmed by quantitative real-time PCR. Down-regulation of miR-186* expression by curcumin elevated the apoptosis, and the survival rate of A549/DDP cells decreased; but up-regulation of miR-186* expression by transfection its mimics restrained the apoptosis, the survival rate of A549/DDP cells increased, which were assayed by flow cytometry expriments and MTT method.

CONCLUSIONModulation of miRNAs expression may be an important mechanism underlying the biological roles of Curcumin.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Curcumin ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; MicroRNAs ; genetics

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Expression of hTERT mRNA in Anip973 and Anip973/NVB cell detected by quantitative real-time PCR.

Gongyan CHEN ; Shuying GUO ; Jingjing LIU

Chinese Journal of Lung Cancer.2010;13(4):297-300. doi:10.3779/j.issn.1009-3419.2010.04.05

BACKGROUND AND OBJECTIVEHuman telomerase reverse transcriptase is the catalytic subunit of telomerase, and its activity is correlated with cell's sensitivity to chemotherapy. The aim of this study is to investigate the differential expression of human telomerase reverse transcriptase (hTERT) mRNA in human lung adenocarcinoma cell line Anip973 and Anip973/NVB, and to observe the correlation between hTERT mRNA and drug-resistance.

METHODSThe real-time fluorescence quantitative RT-PCR was used to detect the change of hTERT mRNA in human lung adenocarcinoma drug-resistant cell Anip973/NVB and parental cell Anip973 treated by NVB.

RESULTSIn the control group, the expression of hTERT mRNA showed no significant difference between drug-resistant cell Anip973/NVB and parental cell Anip973. After been treated by NVB, the expression of hTERT mRNA in parental cell was significantly decreased (P < 0.01), and drug-resistant cell Anip973/ NVB had no evidently variant (P > 0.05). The down-regulated hTERT mRNA in Anip973 cell was higher than that in Anip973/ NVB cell.

CONCLUSIONTelomerase correlates with the drug-resistant cell A973/NVB, and telomerase may be a new target for multi-drug resistant inversion.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; genetics ; physiology ; Humans ; Lung Neoplasms ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; genetics ; Vinblastine ; analogs & derivatives ; pharmacology

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Effect of liposomal transfection of UCH-L1 siRNA on proliferation and apoptosis of lung cancer cell line H157.

Xiaojie QU ; Yanfu WANG

Chinese Journal of Lung Cancer.2010;13(4):292-296. doi:10.3779/j.issn.1009-3419.2010.04.04

BACKGROUND AND OBJECTIVESeveral reports demonstrated that the ubiquitin C terminal hydrolase-L1 (UCH-L1) has been found to be an oncogene in malignant tumors such as esophageal carcinoma, lung cancer and breast cancer. The aim of this study is to explore the effects of liposomal transfection of UCH-L1 siRNA on the proliferation and apoptosis of lung adenocarcinoma cell lines H157.

METHODSUCH-L1 siRNA was synthesized and transfected into H157 cell by liposome. Cell morphological change was observed with microscope, and cell proliferation and apoptosis index detected by flow cytometry, UCH-L1 mRNA expression was determined by RT-PCR and protein level of UCH-L1 was determined by Western blot.

RESULTSFor the H157 cell transfected with siRNA, cell proliferation was inhibited significantly, cell apoptosis appeared obviously, the expression of UCH-L1 mRNA and protein level of UCH-L1 significantly decreased.

CONCLUSIONUCH-L1 siRNA is able to inhibit the proliferation of lung adenocarcinoma cell lines H157 and induce the apoptosis. UCH-L1 might become a new target for lung carcinoma gene therapy.

Apoptosis ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; Genetic Therapy ; Humans ; Liposomes ; chemistry ; Lung Neoplasms ; therapy ; RNA, Small Interfering ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Ubiquitin Thiolesterase ; genetics

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Resveratrol inhibits EGF-induced invasion of human lung adenocarcinoma A549 cells.

Ningyu HUANG ; Hong LU ; Lijun CHANG ; Hongwei ZHANG ; Hao ZHANG ; Guanwu LI

Chinese Journal of Lung Cancer.2010;13(4):287-291. doi:10.3779/j.issn.1009-3419.2010.04.03

BACKGROUND AND OBJECTIVEInvasion and metastasis are the primary causes of death in patients with pulmonary carcinoma. The epidermal growth factor (EGF) stimulates A549 cells invasion greatly through activating ERK and PI3K-Akt signaling pathway. The aim of this study is to elucidate the inhibitory effect of Resveratrol on EGF-induced invasive ability of A549 cells in vitro and explore the molecular mechanism.

METHODSThe cytotoxicity of Resveratrol was evaluated by methyl thiazolyltetrazolium (MTT) assay. Then, the A549 cells were treated with EGF and non-cytotoxic concentration of Resveratrol. The cells' invasion were detected by Boyden chamber assay; MMP-2 activity was determined by gelatine zymography assay; the changes of the related proteins were detected by Western blot.

RESULTSResveratrol was not toxic to A549 cells at the concentration between 0 to 30 microM. The invasion ability of EGF-induced A549 cells was decreased after treatment with 20 microM resveratrol for 24 h, accompanied by the inhibition of MMP-2 secretion. And the levels of p-ERK1/2, PI3K (within 6 h) were suppressed too.

CONCLUSION20 microM Resveratrol inhibits A549 cells' invasion possibly through the suppression of the activation of ERK and PI3K-Akt signaling pathways, subsequently exerting inhibitory effect on MMP-2.

Adenocarcinoma ; metabolism ; Anticarcinogenic Agents ; pharmacology ; Blotting, Western ; Cell Line, Tumor ; Epidermal Growth Factor ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; drug effects ; Stilbenes ; pharmacology

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Identification of candidate genes for lung adenocarcinoma using Toppgene.

Guiping WANG ; Yun YE ; Wenling ZHENG ; Wenli MA

Chinese Journal of Lung Cancer.2010;13(4):282-286. doi:10.3779/j.issn.1009-3419.2010.04.02

BACKGROUND AND OBJECTIVELung adenocarcinoma (AC) is the most common type of lung cancer, however, its mechanism of oncongenesis is still unknown. The aim of this study is to screen candidate genes of lung adenocarcinoma using bioinformatics strategy and elucidate the mechanism of lung adenocarcinoma.

METHODSTwo published microarray data (GSE7670 and GSE10072) was obtained from Gene Expression Omnibus (GEO). Significance analysis of microarrays was performed with the software dchip, and differential expression genes from dchip analysis were defined as "test gene set". Genes correlated with lung adenocarcinoma, obtained by data mining tools genecard and Fable were regarded as "train gene set". Finally, candidate genes of lung adenocarcinoma were screened by the tool "Toppgene".

RESULTSThree hundred and forty-four differential genes were defined as "test gene set", and 277 genes correlated with lung adenocarcinoma were regarded as "train gene set". Thirty-six candidate genes were screened out by Toppgene, among them, 21 genes had nearly no report in cancer. In the following QRT-PCR experiment, CD36, PMAIP1 and FABP4 were down-regulated expression in A549, which coincided with the gene chip.

CONCLUSIONIt is demonstrated that Toppgene is useful in identification of the candidate genes of lung adenocacinoma, which provides the proof for the discovery of the specific disease genes.

Adenocarcinoma ; genetics ; Computational Biology ; Data Mining ; Humans ; Lung Neoplasms ; genetics ; Oligonucleotide Array Sequence Analysis

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Biological characteristics and antitumor activity of CIK cells activated by recombinant human fibronectin for human lung cancer cell lines in vitro.

Shiyong WANG ; Weili DU ; Hui ZHANG ; Tuya WULAN ; Yuan ZHANG ; Ying HE ; Yunfeng YANG ; Sa LIU ; Zhe ZHANG ; Jialing WANG

Chinese Journal of Lung Cancer.2010;13(4):277-281. doi:10.3779/j.issn.1009-3419.2010.04.01

BACKGROUND AND OBJECTIVEThe CIK cell is one of the most important means of the adoptive cellular immunotherapy, and it is a hotspot of which to simplify its culture procedure and to promote its inhibition rate. The aim of this study is to observe the biological function of the CIK cells cultivated by the recombinant human fibronectin (RN) and to establish an effective and simple way of cells expansion.

METHODSWe separated the mononuclear cells (PBMCs) in 50 mL peripheral blood from 10 healthy persons with density gradient centrifugation in the lymphocyte-separating medium, and the PBMCs were divided into two groups, of which were cultivated by RN-introduced and conventional method separately. Then we estimated the proliferation ability, and analyzed the immunologic type, IFN-gamma, IL-4, perforin and granzyme B of them with flow cytometry. Besides that, we tested the inhibition rate of CIKs cells to four kinds of human lung cancer cell lines in vitro by MTT assay.

RESULTSThe RN-induced group had a higher proliferation rate that was 2.0-3.5 times of the conventional group, and there was an obvious statistical difference between the two (P < 0.05). The proliferation rates of CD3+CD16+CD56+T cells in each group were 3 778 and 2 068 times of the initial number, respectively. There was also a higher percentage of CD3+CD8+ T cells in RN-induced group (P < 0.05), while the percentage of CD3+CD4+T cells had no significant statistical difference (P > 0.05). We found a similar inhibition rate of the CIK cells to all this human lung cancer cell lines (P > 0.05). The cells which secreted IFN-gamma increased, while the cells which secreted IL-4 did not. The cells which secreted granzyme B and perforin were positive.

CONCLUSIONIt is an effective and simple way to cultivate the CIK cells with RN, which should be adopted.

CD3 Complex ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; CD56 Antigen ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Cytokine-Induced Killer Cells ; drug effects ; metabolism ; Fibronectins ; genetics ; metabolism ; pharmacology ; Flow Cytometry ; Humans ; Immunotherapy, Adoptive ; methods ; Interleukin-4 ; metabolism ; Lung Neoplasms ; therapy ; Receptors, IgG ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; T-Lymphocytes ; immunology

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