Array analysis for potential biomarker of gemcitabine identification in non-small cell lung cancer cell lines.

Gemcitabine is one of the most widely used drugs for the treatment of advanced Non-small cell lung cancer (NSCLC), but modest objective response rate of patients to gemcitabine makes it necessary to identify novel biomarkers for patients who can benefit from gemcitabine-based therapy and to improve the effect of clinical therapy. In this work, 3 NSCLC cell lines displaying different sensitivities to gemcitabine were applied for mRNA and microRNA (miR) expression chips to figure out the biomarkers for gemcitabine sensitivity. Genes whose expression increased dramatically in sensitive cell lines were mainly enriched in cell adhesion (NRP2, CXCR3, CDK5R1, IL32 and CDH2) and secretory granule (SLC11A1, GP5, CD36 and IGF1), while genes with significantly upregulated expression in resistant cell line were mainly clustered in methylation modification (HIST1H2BF, RAB23 and TP53) and oxidoreductase (TP53I3, CYP27B1 and SOD3). The most intriguing is the activation of Wnt/ß-catenin signaling in gemcitabine resistant NSCLC cell lines. The miR-155, miR-10a, miR-30a, miR-24-2* and miR-30c-2* were upregulated in sensitive cell lines, while expression of miR-200c, miR-203, miR-885-5p, miR-195 and miR-25* was increased in resistant cell line. Genes with significantly altered expression and putatively mediated by the expression-changed miRs were mainly enriched in chromatin assembly (MAF, HLF, BCL2, and IGSF3), anti-apoptosis (BCL2, IGF1 and IKBKB), protein kinase (NRP2, PAK7 and CDK5R1) (all the above genes were upregulated in sensitive cells) and small GTPase mediated signal transduction (GNA13, RAP2A, ARHGAP5 and RAB23, down-regulated in sensitive cells). Our results might provide potential biomarkers for gemcitabine sensitivity prediction and putative targets to overcome gemcitabine resistance in NSCLC patients.

Inhibition of survivin expression and mechanisms of reversing drug-resistance of human lung adenocarcinoma cells by siRNA.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis protein (IAP) family, overexpresses in tumor cells and not expresses in terminally differentiated adult tissues. This study aimed to investigate the effects of survivin-specific siRNA on cell proliferation, apoptosis and chemosensitivity to cisplatin in vitro and in vivo and explore the mechanisms about decreasing expression of survivin in reversing cancer cells resistance to chemotherapeutic drug. METHODS: Survivin-specific siRNA was transfected into A549/DDP cells. The expression of survivin and lung resistance-related protein (LRP) mRNA levels were determined by RT-PCR, chemosensitivity of A549/DDP (cisplatin) cells to cisplatin was determined by MTT assay, and apoptosis and cell cycle were determined by flow cytometry (FCM). The protein expression levels of survivin, LRP, cyclin-D(1), caspase-3 and bcl-2 were determined by Western blotting analyses. The effect of survivin siRNA inhibition on tumor growth was studied in athymic nude mice in vivo. RESULTS: Survivin-specific siRNA efficiently down-regulated survivin expression. The cell cycle was arrested at G2/M phase, and apoptosis was obviously found. Inhibition of survivin expression could make the IC50 and drug-resistant index of cisplatin decrease, and enhance the cancer cells sensitivity to cisplatin. After transfection by survivin-specific siRNA, expression of LRP and cyclin-D1 were downregulated, caspase-3 expression was upregulated, bcl-2 expression had no obvious change. The animal experiment confirmed knockdown of survivin could inhibit the tumor growth. CONCLUSIONS: Survivin-specific siRNA can efficiently suppress the expression of survivin, increase apoptosis, inhibit cells proliferation and enhance the chemosensitivity to cisplatin in vitro and in vivo. Suppression of survivin expression helping to reverse drug-resistance may have relationship with downregulation of LRP and upregulation of caspase-3. Anti-tumor strategies based on the inhibition of survivin may be useful in targeting lung adenocarcinomas.

[Relationships between the expressions of intercellular adhesion molecule-1 and tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9 in lung tissues of patients with chronic obstructive pulmonary disease].

OBJECTIVE: To evaluate the correlation between the expressions of intercellular adhesion molecule-1 (ICAM-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9) in lung tissues of patients with COPD. METHODS: Lung tissues from patients with COPD (COPD group, n = 19) and those without COPD (smokers and nonsmokers with normal lung function, n = 11 and 9, respectively) were obtained from surgical excisions of lung cancer patients. The mRNA expression of ICAM-1, TIMP-1 and MMP-9 was detected using semi-quantitative RT-PCR. The protein expression of ICAM-1, TIMP-1 and MMP-9 was detected by using immunohistochemistry method. RESULTS: There were significant differences in FEV1% and FEV1/FVC% among smokers without COPD, nonsmokers without COPD and COPD patients. MMP-9 was highly expressed in alveolar epithelial cells, bronchial epithelial cells, vascular smooth muscle cells, alveolar macrophages, and interstitial cells in the COPD group, compared with smokers without COPD group and nonsmokers without COPD group (54.0 +/- 15.0), (1.2 +/- 0.7) and (1.4 +/- 0.8). Low level expression of TIMP-1 was detected in alveolar macrophages, alveolar epithelial cells and vascular smooth muscle cells in the COPD group, but no expression in smokers and nonsmokers without COPD. High level expression of ICAM-1 was detected in alveolar epithelial cells, and the expression was higher in the COPD group (52.1 +/- 13.4), (2.1 +/- 1.1) and (4.5 +/- 2.4). The mRNA level of MMP-9 showed significant difference among patients with COPD, smokers without COPD and nonsmokers without COPD (0.71 +/- 0.16), (0.20 +/- 0.08) and (0.17 +/- 0.05). The mRNA level of TIMP-1 was also significantly different among patients with COPD, smokers without COPD and nonsmokers without COPD (0.47 +/- 0.10), (0.26 +/- 0.08) and (0.20 +/- 0.06). ICAM expression was also significantly higher in patients with COPD as compared with smokers without COPD and nonsmokers without COPD (0.62 +/- 0.15), (0.44 +/- 0.12) and (0.37 +/- 0.11). Both the mRNA and the protein levels of MMP-9 were inversely correlated with FEV1 % and FEV1/FVC% (r= -0.759, -0.756, -0.772, -0.725, respectively, P <0.01). TIMP-1 mRNA level was inversely correlated with FEV1% and FEV1/FVC% (r = -0.675, -0.623, respectively P <0.01). Negative correlations were also noted between ICAM-1 expressions (both mRNA and protein) and FEV1% or FEV1/FVC% (r = -0.580, -0.531, -0.739, -0.756, respectively P <0.01). Interestingly, the mRNA expression of TIMP-1, MMP-9 and ICAM-1 was positively correlated (r = 0.576, 0.524, P < 0.01), while the protein levels of MMP-9 and ICAM-1 were positively correlated (r = 0.964, P <0.01). CONCLUSION: There was a significant correlation between over-expression of ICAM-1 and TIMP-land MMP-9 in lung tissues from COPD patients. Over-expressions of ICAM-1 in the lung may result in accumulation of inflammatory cells releasing certain inflammatory factors that could destroy the normal lung structure. In addition, highly expressed TIMP-1 and MMP-9 in lung tissues may also contribute to the destruction and reconstitution of the bronchial or/and alveolar wall, which is likely to play a major role in airway obstruction.

[Expressions of and the effect of smoking on vascular endothelial growth factor and induced nitric oxide synthase in lung tissues of chronic obstructive pulmonary disease patients].

OBJECTIVE: To investigate the expressions of and the effect of smoking on vascular endothelial growth factor (VEGF) and induced nitric oxide synthase (iNOS) in lung tissues of chronic obstructive pulmonary disease (COPD) patients. METHODS: The peripheral lung tissues were obtained from 46 patients with lung carcinoma. They were divided into three groups according to their habit of smoking and lung function, 19 smokers with moderate COPD, 12 smokers and 15 nonsmokers with normal lung function. The expression of VEGF and iNOS was detected by immunohistochemistry. RESULTS: Expressions of VEGF and iNOS were increased in lung tissues of smokers without COPD (1.50 +/- 0.39, 1.45 +/- 0.41) compared with nonsmokers without COPD (1.18 +/- 0.33, 1.09 +/- 0.41) (each P < 0.05), and were significantly increased in lung tissues of smokers with moderate COPD (2.19 +/- 0.51, 2.39 +/- 0.45) compared with nonsmokers without COPD (each P < 0.01). The expression of VEGF in lung tissues was significantly correlated with the expression of iNOS (r = 0.78, P < 0.01), but was inversely correlated with FEV(1) (r = -0.67, P < 0.01). CONCLUSIONS: Expressions of VEGF and iNOS were upregulated in lung tissues of smokers and patients with moderate COPD. Overexpression of iNOS and VEGF may participate in the mechanism of airway and vascular remodeling, and airflow limitation in COPD.

[Analysis of gene expression profile in lung adenocarcinoma, tumor adjacent tissue and fetal lung tissue].

OBJECTIVE: To investigate the gene expression profiles in lung adenocarcinoma (LA), tumor adjacent tissue (TAT) and fetal lung tissue (FLT) by cDNA microarray technique. METHODS: Total RNA from LA, TAT and FLT was extracted and purified. The cDNA was made by RT-PCR, and then labeled with Cy5 and Cy3 fluorescence as probes which were hybridized with the whole gene chips. Subsequently, the signal images were scanned by ScanArray 4000 fluorescence scanner and analyzed by Gene Pix PRO3.0. RESULTS: In 4 cases with LA and TAT, 25 genes were screened out for differences in gene expression level, among which 3 were upregulated and 22 downregulated; in FLT and TAT cases, 316 genes were screened out, among which 192 were upregulated and 124 downregulated; 16 genes were found to be differentially expressed genes in common in LA, TAT and FLT, among which 12 were upregulated and 4 downregulated. CONCLUSION: The 25 differentially expressed genes in LA and TAT may be related to occurrence and development of lung cancer, while the 316 genes in FLT and TAT may be related to fetal developmental. The 16 differentially expressed genes may be related to the initiation of lung cancer.