Proteome analysis and tissue array for profiling protein markers associated with type B thymoma subclassification.

BACKGROUND: The prognostic relevance of World Health Organization (WHO) subtypes within type B thymomas is still controversial. Understanding of the molecular characteristics of the different histologic types of thymomas will provide meaningful information for diagnosis and therapeutic management in type B thymoma. METHODS: Proteins extracted from twelve type B thymoma tissue specimens (six type B1 and six type B2) were analyzed by two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF-MS. Differentially expressed proteins were then assayed in sixty-nine type B thymoma tissues (including B1, B2 and B3) by tissue array analysis with immunohistochemistry staining. The relationship of their expression with clinicopathological parameters, such as tumor stage or WHO classification, was estimated by Spearman's Rank Correlation Test. RESULTS: Sixteen differentially expressed proteins between type B1 and B2 thymoma tissues were identified. The differential levels of ezrin and glutathione S-transferase pi (GSTP1) were validated using immunohistochemistry staining. A statistically significant difference was observed in the positive rate of ezrin expression between type B1 thymoma and type B3 thymoma (Z = -2.963, P < 0.01). Ezrin showed a tendency to be expressed in higher classification tumors from type B1 to B3. A statistical analysis demonstrated that type B2 and B3 tumors had significantly higher positive expression of GSTP1 than the B1 group (type B2 vs. B1: Z = -2.582, P = 0.01; type B3 vs. B1: Z = -4.012, P ≤ 0.001). The results also showed a strong correlation between GSTP1 and WHO type staging of B1 to B3 tumors (Spearman's correlation coefficient: 0.633, P ≤ 0.001). Statistical analysis showed that there was close correlation between GSTP1 and ezrin expression with the clinical stage (Spearman's correlation coefficients, ezrin: 0.481, P < 0.05; GSTP1: 0.484, P < 0.01). CONCLUSIONS: Differentially expressed proteins between type B1 and B2 thymoma tissues were analyzed by comparative proteomic analysis. The techniques of proteomic analysis and tissue array provide a potential tool for screening of key molecules in type B thymoma histological sub-classifications. The statistical analysis of ezrin and GSTP1 expression by immunohistochemistry, especially GSTP1, may be a useful approach for type B thymoma classification.

Expression of GPC3 protein and its significance in lung squamous cell carcinoma.

The purpose of this study was to investigate GPC3 gene expression in lung squamous cell carcinoma tissue and its correlation with clinical and tumor characteristics. Using RT-PCR, the presence of GPC3 gene expression was detected in cancer tissue and adjacent normal tissue in 66 cases of lung squamous cell carcinoma and positive rates were calculated. Using Western blot, changes in GPC3 protein expression were detected in lung squamous cell carcinoma and adjacent normal tissues. The percentage of tissue samples expressing GPC3 mRNA was significantly higher in lung squamous cell carcinoma than in adjacent normal tissue (P < 0.05). This percentage was also significantly higher for cases with lymph node metastasis than for those without lymph node metastasis (P < 0.05). Further, the percentage of samples expressing GPC3 mRNA was higher with lowering degrees of tumor differentiation (P < 0.05). Rates of GPC3 expression were, however, independent of patient gender, age, and tumor size (P > 0.05). The expression of GPC3 protein in lung squamous cell carcinoma was significantly higher than that in adjacent normal tissues (P < 0.05). The expression in cases with lymph node metastasis was significantly higher than in those without lymph node metastasis (P < 0.05), and GPC3 protein expression increased with lowering degrees of tumor differentiation (P < 0.05). Further investigation is warranted for the association of initiation, development, invasion, and metastasis of disease.

Immunomodulation of inducible co-stimulator (ICOS) in human cytokine-induced killer cells against cholangiocarcinoma through ICOS/ICOS ligand interaction.

OBJECTIVE: To evaluate the immunomodulation of inducible co-stimulator (ICOS) in cytokine-induced killer (CIK) cells against cholangiocarcinoma. METHODS: CIK cells were generated from normal peripheral blood mononuclear cells. Methyl thiazolyl tetrazolium assay was performed to assess proliferation of CIK-ICOS and controlled CIK cells; ELISA was used to analyze the expression of cytokines. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of ICOS ligand (ICOSL) in CIK cells and human cholangiocarcinoma cell line QBC939 cells. The cytotoxicity of CIK cells was determined either by lactate dehydrogenase-releasing assay in vivo or alteration of tumor size prior to and after the treatment of CIK cells in vivo. RESULTS: CIK-ICOS cells proliferated more and expressed higher secretion a level of interferon-γ than the controlled CIK. These cells exhibited higher cytotoxicity against cholangiocarcinoma cell lines at all efficacy: toxicity (E:T) ratios tested than the controlled CIK cells. More importantly, the anti-ICOSL antibody was able to attenuate the elevated cytotoxicity mediated by ICOS overexpression. When injected into cholangiocarcinoma xenografts in severe combined immunodeficiency mice, CIK-ICOS cells survived better than the controlled CIK cells around xenografts and significantly reduced the growth rate of cholangiocarcinoma, with least volume increase and more severe necrosis of the xenografts than controlled mice treated with saline, CIK or CIK-enhanced green fluorescent protein. CONCLUSION: ICOS can enhance the cytotoxic effect of CIK cells against cholangiocarcinoma both in vitro and in vivo. This effect is mediated by ICOS-augmented cytokine secretion and cell proliferation, and in part through ICOS-ICOSL interaction.

Comparative proteomic analysis of paclitaxel sensitive A549 lung adenocarcinoma cell line and its resistant counterpart A549-Taxol.

PURPOSE: Paclitaxel is used as the first-line chemotherapy for Non-Small Cell Lung Cancer (NSCLC), but acquired resistance becomes a critical problem. Several mechanisms have been proposed in paclitaxel resistance, but they are not sufficient to exhaustively explain this resistance emergence. To better investigate molecular resistance mechanisms, a comparative proteomic approach was carried out to identify differentially expressed proteins between human lung adenocarcinoma A549 cell line (paclitaxel sensitive) and A549-Taxol cell line (acquired resistant). METHODS: A paclitaxel-resistant subline (A549-Taxol) derived from the parental-sensitive cell line A549 was established by stepwise selection by paclitaxel. Total proteins in the two cell lines were separated by fluorescent differential gel electrophoresis (DIGE). Image analysis was carried out with the DeCyder 2D 6.5 software. Proteins associated with chemoresistance process were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Some key molecules were valuated by Western blot. RESULTS: Thirty proteins were identified and grouped into eight main functional classes according to the biological processes in which they are likely to participate, i.e. signal transduction, cytoskeleton, redox reaction, energy and metabolism, and so on. Alterations of these processes might be involved in paclitaxel resistance. Most of the proteins showed mitochondrial and cytoplasm location. The up-regulation of CK8, CK18, ALDH1, CAST and ANX I in A549-Taxol cell line was verified by Western blot, in coincidence with the data obtained from proteomic analysis. CONCLUSION: For the first time, differentially expressed proteins between paclitaxel-sensitive cell line and paclitaxel-resistant one were explored by comparative proteomic approach in human lung adenocarcinoma. It may be useful for further studying of resistance mechanisms and screening of resistance biomarkers, so as to develop tailored therapeutic strategies.

[The application of endobronchial ultrasound-guided transbronchial needle aspiration: report of 70 cases].

OBJECTIVE: To evaluate the diagnostic yield and the safety of endobronchial ultrasound-guided transbronchial needle biopsy (EBUS-TBNA) in mediastinal and hilar lymph nodes and lung tumors. METHODS: EBUS-TBNA was performed in 70 patients with thoracic masses or mediastinal-hilar lymphoadenopathy proved by CT scan. RESULTS: From July 2009 to January 2010, 70 patients were included in the study. EBUS-guided TBNA was performed to obtain samples from mediastinal and hilar lymph nodes (120 stations) and lung tumors (11 masses). In 46 cases of newly diagnosed lung cancer, 44 were confirmed by EBUS-TBNA without on site cytology assistance, with 2 false negative cases. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of EBUS-TBNA in the diagnosis of lung cancer were 96%, 100%, 100%, 92% and 97% respectively. Non-caseous granuloma formed by epithelioid cells was found in EBUS-TBNA histological specimen from 5 out of 10 patients with clinically diagnosed sarcoidosis. TBNA cytological smear showed acid-fast bacilli and histology of the lymph node demonstrated coagulatory necrosis from 1 out of 4 tuberculous cases. The procedure was uneventful, and there were no complications. CONCLUSION: EBUS-TBNA is an effective and safe method for the diagnosis of bronchogenic carcinoma and unknown mediastinal-hilar lymphadenopathy.

The expression of stem cell-related indicators as a prognostic factor in human lung adenocarcinoma.

INTRODUCTION: The purpose of the present study was to detect the presence of BASC-like stem cell-related indicators, such as clara cell secretory protein (CCSP), Octamer-4 (OCT4) and Bmi-1, and evaluate their implications in the prognosis of patients with lung adenocarcinoma. METHODS: Specimens of 134 cases of lung adenocarcinoma were collected after radical surgery from January 1999 to June 2004. RESULTS: One hundred and twenty-six cases showed cells that were positive for CCSP, 99 cases positive for OCT4, 91 cases simultaneous expression of CCSP and OCT4 and 74 cases positive for Bmi-1. Bmi-1 was significantly higher in patients at stage III compared to patients at stages I and II. The pattern of survival curves showed that Bmi-1 was a significant prognostic factor of poor overall survival in lung adenocarcinoma patients (P = 0.0000), and the patients with OCT4(+) expression showed a greater increase in mortality than OCT4(-) patients (P = 0.0103). The results of univariate and multivariate Cox analysis revealed that the pathological stages of tumor node metastases (P = 0.037), OCT4 (P = 0.046) and Bmi-1 expression (P = 0.001) were independent prognostic factors. CONCLUSIONS: OCT4 and Bmi-1 may be good biomarkers to predict the prognosis of patients with completely resected lung adenocarcinoma.

Discrepancy of biologic behavior influenced by bone marrow derived cells in lung cancer.

Disseminated cancer cells may initially require local nutrients and growth factors to thrive and survive in bone marrow. However, data on the influence of bone marrow derived cells (BMDC, also called bone stromal cells in some publications) on lung cancer cells is largely unexplored. This study explored the mechanism of how bone stromal factors contribute to the bone tropism in lung cancer. The difference among lung cancer cell lines in their abilities to metastasize to bone was found using the SCID animal model. Supernatant of bone marrow aspiration (BM) and condition medium from human bone stromal cells (BSC) were used to study the activity of bone stromal factors. We found bone stromal factors significantly increased the proliferation, invasion, adhesion and expression of angiogenosis-related factors, and inhibited the apoptosis for high bone metastasis H460 lung cancer cells. These biologic effects were not seen in SPC-A1 or A549 cells, which are low bone metastasis lung cancer cells. Adhesion of H460 cells to surface coated with bone stromal cells can activate some signal transduction pathways, and alter the expression of adhesion associated factors, including integrin ß 3 and ADAMTS-1, two potential targets related with bone metastasis. We concluded that bone marrow derived cells had a profound effect on biological behavior of lung cancers, therefore favoring the growth of lung cancer cells in bone.

[Effects of a protein kinase C inhibitor combined with cisplatin on non-small cell lung cancer].

OBJECTIVE: To study the effects and possible mechanisms of the protein kinase C (PKC) inhibitor chelerythrine chloride (CH), combined with cisplatin (DDP) on human non-small cell lung cancer. METHODS: The effect of CH, DDP and the combination on proliferation and apoptosis of human lung cancer cell line A549 were evaluated by MTT assay and flow cytometry respectively. The inhibitory effects of CH and DDP on neoplasia were verified on subcutaneous implanted tumor of nude mice. Implanted tumor models were constructed in nude mice using human lung adenocarcinoma cell line A549. Twenty-four BALB/c nude mice with implanted tumors were divided into 4 groups randomly: group CH, group DDP, group CH + DDP, and normal saline group (group NS), each with 5 mice. CH, DDP or NS were intraperitoneally injected into nude mice for 3 weeks (DDP or NS was injected once a week, CH was injected twice a week). RESULTS: CH inhibited A549 cell proliferation in a concentration-dependent pattern. When CH and DDP were combined, the inhibitory effect was enhanced in a synergistic or additive pattern. Both CH and DDP significantly increased the apoptosis of A549 cells, and this action was remarkably increased when DDP was combined with CH. CH and DDP inhibited the growth of subcutaneous implanted tumor in nude mice and the inhibitory rate of group CH + DDP (80.5%) was significantly higher than that of group CH (72.4%) or group DDP (64.3%) (t = 11.34, P < 0.01). CONCLUSION: CH combined with DDP shows significantly synergistic anti-tumor effects on non-small cell lung cancer cell line A549 and subcutaneous implanted tumor in nude mice, possibly by enhancement of growth inhibition and apoptosis induction on tumor cells.

Prognostic impact of vascular endothelial growth factor-A and E-cadherin expression in completely resected pathologic stage I non-small cell lung cancer.

OBJECTIVE: The purpose of this study was to evaluate the value of vascular endothelial growth factor-A and E-cadherin expression as well as other confirmed prognostic factors in predicting the clinical outcome after definitive surgery of pathologic stage I non-small cell lung cancer. METHODS: One hundred and eighty-five consecutive and non-selected patients who underwent definitive surgery for stage I non-small cell lung cancer in our institute were included in this study. Formalin-fixed paraffin-embedded specimens were stained for vascular endothelial growth factor-A and E-cadherin and the correlation between the staining, its clinicopathological parameters and its prognostic power were analyzed statistically. RESULTS: Of the 185 patients studied, 92 cases (49.7%) were strongly positive for vascular endothelial growth factor-A. Vascular endothelial growth factor-A expression was only related to visceral pleural involvement (P < 0.001). A total of 95 carcinomas (51.4%) were E-cadherin-negative tumors. E-cadherin expression correlated with histology (P < 0.001), tumor size (P = 0.001) and visceral pleural involvement (P < 0.001). In univariate analysis by log-rank test, gender, tumor size, lymphovascular invasion, visceral pleural involvement, vascular endothelial growth factor-A expression and E-cadherin expression were significant prognostic factors (P = 0.003, 0.042, 0.026, 0.035, 0.008 and 0.006, respectively). In multivariate analysis, gender, vascular endothelial growth factor-A and E-cadherin expression maintained its independent prognostic influence on overall survival (P = 0.013, <0.001 and 0.036, respectively). CONCLUSIONS: Expression of vascular endothelial growth factor-A is related to visceral pleural involvement, and E-cadherin expression correlates with histology, tumor size and visceral pleural involvement. Multivariate analysis confirmed gender, vascular endothelial growth factor-A and E-cadherin expression were significant predictive factors for overall survival in completely resected pathologic stage I non-small cell lung cancer.

[Dual regulation of cell cycles by Shuanghuang Shengbai Granule in Lewis-bearing mice with chemotherapy-induced myelosuppression and its mechanism].

OBJECTIVE: To explore the dual regulatory effects of Shuanghuang Shengbai Granule, a compound traditional Chinese herbal medicine, on cell cycle in Lewis-bearing mice with chemotherapy-induced myelosuppression. METHODS: Thrity Lewis-bearing mice were randomly divided into control group, untreated group and treated group. A model of myelosuppression was established by peritoneal injection of cyclophosphamide to Lewis-bearing mice. Mice in the treated group were treated with Shuanghaung Shengbai Granule for 6 days. Cell cycle progressions of cells collected from bone marrow and tumor tissues were assayed by flow cytometry, and proliferation index (PI) was also calculated. Expressions of cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6) and cyclin D1 in bone marrow and tumor tissues were detected by Western blotting and immunohistochemical method. RESULTS: Percentages of bone marrow and tumor cells in G0/G1 phase in the untreated group were lower than those in the control group; however, the PI of untreated group was higher than that of the control group. The expressions of CDK4, CDK6 and cyclin D1 in bone marrow and tumor tissues in the untreated group were increased as compared with the control group. Compared with the untreated group and the control group, the percentage of bone marrow cells in G0/G1 phase was decreased, and the PI of bone marrow cells and the expressions of CDK4, CDK6 and cyclin D1 were increased in bone marrow in the treated group. However, the percentage of tumor cells in G0/G1 phase in the treated group was increased, and the PI of tumor cells and the expressions of CDK4, CDK6 and cyclin D1 in tumor tissues were decreased as compared with the untreated and control groups. CONCLUSION: Shuanghuang Shengbai Granule may have a function of cell cycle dual regulation in Lewis-bearing mice with chemotherapy-induced myelosuppression by regulating the expressions of CDK4, CDK6 and cyclin D1 in bone marrow and tumor tissues.

[Efficient generation and functional characteristics of dendritic cells from malignant pleural effusion in patients with lung cancer].

OBJECTIVE: To clarify whether functionally competent dendritic cells (DC) can be generated from malignant pleural effusion in patients with lung cancer. METHODS: Malignant effusion-associated monocytes were separated by adherence from malignant effusion-associated mononuclear cells and cultured in medium with granulocyte macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4). TNF-alpha was added for the last 24 h before culture termination. Cultured DC were identified by (1) using microscopy, scanning electron microscopy, and immunocytochemistry for the morphological features; (2) phenotypic markers; and (3) functional characteristics including a high stimulatory capacity to activate proliferation of lymphocyte in an allogeneic mixed leukocyte reaction and the ability to produce high levels of IFN-gamma. RESULTS: Cultured DC had the typical morphological features. The phenotype of DCs generated from effusion showed higher expression of CD(86) (84.6 +/- 6.1)%, HLA-DR (81.1 +/- 13.0)%, CD(40) (42.0 +/- 21.7)%, CD(1a) (20.0 +/- 9.5)% and lower expression of CD(14) (4.8 +/- 3.5)% than the control group. There was a significant difference in the stimulatory activity in allogeneic lymphocyte proliferation and the ability to produce high levels of IFN-gamma between DC derived from the malignant effusion and the control group. CONCLUSION: These findings suggest that DC can be generated from malignant pleural effusion, which might be a useful source of DC for immunotherapy.

[Experimental study of Qiyeling Decoction inducing apoptosis of transplanted human lung adenocarcinoma cells A549 in nude mice].

OBJECTIVE: To study the function of Qiyeling Decoction in inducing apoptosis of transplanted human lung adenocarcinoma cells A549 in nude mice. METHODS: Nude mice with transplanted A549 tumor were randomly divided into the untreated control group (group A), chemotherapy treated group (group B), chemotherapy plus Qiyeling Decoction treated group (group C), Qiyeling Decoction treated group (group D) and managed correspondingly. The tumor volume was measured and calculated into tumor weight. The apoptosis of tumor cells were examined using in situ cell apoptosis detection kit. RESULTS: The tumor weight was lower obviously in groups B, C and D than that in group A (P<0.05). The apoptosis of tumor cells was lower obviously in groups B, and C than that in group D (P<0.05). Cells in group A appeared perfect differentiation during the early stage and apoptosis later. CONCLUSION: Qiyeling Decoction can induce A549 cell apoptosis in nude mice.

Human acyl-coenzyme A:cholesterol acyltransferase 1 (acat1) sequences located in two different chromosomes (7 and 1) are required to produce a novel ACAT1 isoenzyme with additional sequence at the N terminus.

A rare form of human ACAT1 mRNA, containing the optional long 5'-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7. To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5'-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1(ORF). The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

[Vascular endothelial growth factor inhibits dendritic cells from patients with non-small cell lung carcinoma].

OBJECTIVE: To detect the effect of vascular endothelial growth factor (VEGF) on dendritic cells (DC) in patients with non-small cell lung cancer (NSCLC). METHODS: The measurement of DC in the peripheral blood was performed by a novel flow cytometric assay in 85 patients with NSCLC and 14 healthy volunteers. Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration of VEGF(165) in the plasma. CD(14)(+) peripheral blood mononuclear cells (PBMC) were cultured to obtain DC in vitro with cytokines. VEGF(165) was added to evaluate its effect on DC differentiation and survival. The phenotypes and apoptosis of cultured cells were detected by flow cytometry. RESULTS: In comparison with healthy volunteers, the level of VEGF(165) was significantly increased (P < 0.05), while that of DC was significantly decreased (P < 0.01) in patients with NSCLC. No significant correlation was noted between the concentration of VEGF(165) and age, gender, differentiation and histological types in patients with NSCLC, neither was found in the level of DC (P > 0.05). The concentration of VEGF(165) was closely associated with TNM stage and distal metastasis (P < 0.05), while no correlation was found between the concentration of VEGF(165) and lymph node metastasis (P > 0.05). Significant correlations were noted between the level of DC in patients with NSCLC and TNM stage, lymph node metastasis and distal metastasis (P < 0.05). There was a negative correlation between the concentration of VEGF(165) and the level of DC (P < 0.05). Patients with abnormally elevated VEGF(165) showed significantly fewer DC. Cells cultured in vitro in the presence of VEGF(165) exhibited higher expression of CD(+)(14)(P = 0.000) and increased ratio of apoptic cells (P < 0.01), but decreased expression of CD(40), CD(86) and HLA-DR (P < 0.01), as compared to cells cultured without VEGF(165). CONCLUSIONS: The level of DC and the concentration of VEGF in the peripheral blood can reflect the malignancy of NSCLC. NSCLC can over-express VEGF to inhibit DC differentiation and maturation to evade host immune surveillance.