Non-neoplastic Lung Tissue Research Articles

P1.11 Stage-Associated Difference in Microrna Expression in Lung Adenocarcinoma from Smokers and Non-Smokers

ABSTRACT Background To identify alterations in miRNA expression that occur in the early and late stages of lung cancer in smokers and non-smokers, we conducted miRNA profiling analysis. MiRNAs abnormally expressed in lung adenocarcinomas were compared in smokers vs. non-smokers as well as early stage vs. late stage disease. Materials and methods We collected 62 lung adenocarcinoma samples with paired non-neoplastic lung tissue from patients with no pre-operative therapy at Fox Chase Cancer Center and Methodist Hospital. Twenty-five patients had no history of tobacco smoking. The clinical stages were proportionally matched between smokers and non-smokers. MiRNA expression was profiled using the Human miRNA V2 Oligo Microarray Kit (Agilent Technologies, Alto, CA). The human miRNAs were background-subtracted, quantile-normalized in arrays, and log-2 transformed. The differences of mRNAs between tumor and normal tissue were examined using two-factor linear model. We assessed the local false discovery rates (FDRs) for all terms including the intercept using the Bum method (Pounds and Morris, 2003). Results We identified 63 miRNAs with FDR Table 1 . Conclusion Eighty-one percent of the altered miRNAs were observed in both smokers and non-smokers, which suggests a common epigenetic predisposition for lung adenocarcinoma development. Altered miRNAs expressed in early stage lung adenocarcinoma may be used as markers for the early detection of lung cancer. Table 1 . MiRNAs abnormally expressed in lung adenocarcinoma Patient characteristics miRNAs S/N/E/L 126, 130a, 135b, 141, 144, 145, 21, 21*, 200b, 210, 218, 30a, 30a*, 30c, 338-3p, 424, 451, 486-5p, 494, 497, 574-5p, 96, 99a S/N/E 200, 429 N/E/L 34c-5p N/L 135a*, 198 S, smoker; N, non-smoker; E, early stage; L, late stage.

MiRNAs expression profiling to distinguish lung squamous-cell carcinoma from adenocarcinoma subtypes

We investigated whether miRNA expression profiles can distinguish and predict outcome of non-small-cell lung carcinoma (NSCLC) patients with different histological subtypes. High-throughput microarray was used to measure miRNA expression levels in six NSCLC samples. Subsequently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify findings in an independent set of 54 squamous-cell lung carcinomas (SCC), 51 lung adenocarcinomas (AD), and paired adjacent non-neoplastic lung tissue. We showed that, compared to adjacent non-neoplastic lung tissues, the expressions of miR-125a-5p and let-7e were decreased in AD and SCC samples, while increased expressions of miR-93, miR-205, and miR-221 were observed in SCC samples. In addition, miR-205 expression was significantly higher in SCC patients with lymph node metastasis. Lower let-7e expression was associated with lymph node metastasis, >3 cm tumor size, and differentiation of the NSCLC AD subtype. High levels of miR-100 expression also correlated with the AD subtype in current smokers. Moreover, induction of miR-93 and miR-205 expressions and reduction of let-7e were strongly associated with shorter overall survival in SCC patients, whereas AD patient survival was only associated with reduced let-7e. We identified differential expression profiles of miRNAs in AD and SCC. More importantly, in addition to morphology and immunocytochemistry approaches, we report that miR-93, miR-205, miR-221, and let-7e may represent novel biomarkers for differential diagnosis and prognosis of certain NSCLC subtypes or be new targets of histology-specific treatments. Furthermore, our results suggest a strong correlation between high expression of miR-100 and AD patients with history of heavy smoking.

Overexpression of Periostin Protein in Non-Small Cell Lung Carcinoma is Not Related with Clinical Prognostic Significance

Background: Periostin is preferentially expressed in periosteum, indicating a potential role in bone formation. Recently, there have been emerging controversies about its role in invasion and metastasis of human malignancies. We attempted to determine the clinicopathological significance of periostin expression in non-small cell lung carcinoma (NSCLC). Methods: Immunohistochemical staining of periostin protein from 91 cases of NSCLCs was performed using tissue microarray blocks. The results were correlated with clinicopathological parameters. Results: Positive reaction to periostin was predominantly noted in the tumor stroma. The strongest reaction presented as a band-like pattern just around the tumor nests. Non-neoplastic lung tissue and most in-situ carcinomas did not show a positive reaction in their stroma. With respect to tumor differentiation, moderate to poor differentiated tumors (47/77) revealed even higher periostin expression than the well-differentiated ones (4/14) (p=0.024). High periostin expression was positively correlated with E-cadherin and p53 expression, but was not related with patient age, sex, tumor type, PCNA index, b-catenin, cyclin D1, pTNM-T, pTNM-N, stage, and patient survival (p>0.05). Conclusion: These results suggest that periostin might play a role during the biological progression of NSCLC, but may not be related to the clinical prognostic parameters.

Bayesian probit regression model for the diagnosis of pulmonary fibrosis: proof-of-principle

BackgroundThe accurate diagnosis of idiopathic pulmonary fibrosis (IPF) is a major clinical challenge. We developed a model to diagnose IPF by applying Bayesian probit regression (BPR) modelling to gene expression profiles of whole lung tissue.MethodsWhole lung tissue was obtained from patients with idiopathic pulmonary fibrosis (IPF) undergoing surgical lung biopsy or lung transplantation. Controls were obtained from normal organ donors. We performed cluster analyses to explore differences in our dataset. No significant difference was found between samples obtained from different lobes of the same patient. A significant difference was found between samples obtained at biopsy versus explant. Following preliminary analysis of the complete dataset, we selected three subsets for the development of diagnostic gene signatures: the first signature was developed from all IPF samples (as compared to controls); the second signature was developed from the subset of IPF samples obtained at biopsy; the third signature was developed from IPF explants. To assess the validity of each signature, we used an independent cohort of IPF and normal samples. Each signature was used to predict phenotype (IPF versus normal) in samples from the validation cohort. We compared the models' predictions to the true phenotype of each validation sample, and then calculated sensitivity, specificity and accuracy.ResultsSurprisingly, we found that all three signatures were reasonably valid predictors of diagnosis, with small differences in test sensitivity, specificity and overall accuracy.ConclusionsThis study represents the first use of BPR on whole lung tissue; previously, BPR was primarily used to develop predictive models for cancer. This also represents the first report of an independently validated IPF gene expression signature. In summary, BPR is a promising tool for the development of gene expression signatures from non-neoplastic lung tissue. In the future, BPR might be used to develop definitive diagnostic gene signatures for IPF, prognostic gene signatures for IPF or gene signatures for other non-neoplastic lung disorders such as bronchiolitis obliterans.

Promoter methylation of ASPP1 and ASPP2 genes in non-small cell lung cancers

To investigate the methylation status of CpG islands in the promoter region and the protein expression of ASPP1 and ASPP2 genes in non-small-cell lung cancer (NSCLC) and their relationship with cellular apoptosis and p53 gene expression. The 5'CpG island methylation patterns of ASPP1 and ASPP2 were evaluated by methylation specific polymerase chain reaction (MSP) followed by confirmation of sequencing. Immunohistochemistry was used to detect the expression of ASPP1, ASPP2 and p53 in lung carcinoma tissue samples (n = 90) and adjacent non-neoplastic lung tissue samples (n = 25). TUNEL assay was used to detect the apoptotic activity. The presence of ASPP1 methylation was significantly higher in NSCLC than that in the adjacent non-neoplastic lung tissue [42.2% (38/90) vs. 16.0% (4/25), P = 0.019]. ASPP1 promoter methylation had a close relationship with TNM stage and lymph node metastasis (P = 0.031, P = 0.030), but was not related to the age, sex, histological types and the grades of tumor differentiation (P = 0.389, P = 0.278, P = 0.570, and P = 0.103). Tumors with ASPP1 promoter methylation demonstrated a lower expression of ASPP1 as compared with those without the methylation (P = 0.002). ASPP1 expression was associated with a higher apoptotic index (AI) (P = 0.022) and a decreased p53 expression (r = -0.259, P < 0.01). Methylation in the promoter region of ASPP2 gene was not detected in lung cancer (n = 90) or adjacent non-neoplastic lung tissue (n = 25). Expression of ASPP2 protein did not correlate with AI (P = 0.282) and p53 status in NSCLC. High methylation of ASPP1 gene promoter regions is one of the important mechanisms that down-regulate its protein expression in NSCLC. ASPP1 promoter methylation may be associated with the malignant progression of the tumor, and ASPP1 expression promotes cellular apoptosis.

Activity-based proteomics: Identification of ABHD11 and ESD activities as potential biomarkers for human lung adenocarcinoma

Lung cancer is the leading cause of all cancer related deaths with a worldwide mortality of 1.2 million each year. The 5-year survival rate ranges from 80% in early stages to a dismal 5% in advanced disease. Prognosis is currently mostly determined based on the extension of disease at diagnosis. Thereby it has become evident that predicted and real outcomes can vary significantly, even for patients with the same stage of disease. Novel biomarkers with a reliable predictive significance are therefore clearly needed. In this study we implemented an activity-based, solely mass spectrometry dependent biomarker discovery platform. We investigated the role of serine hydrolase activities as potential biomarkers for human lung adenocarcinoma, the most common lung cancer subtype. Forty pairs of fresh frozen malignant and matching non-neoplastic lung tissues were analyzed and enzymatic activities linked to clinical follow-up data. We found that the activities of Abhydrolase domain-containing protein 11 and Esterase D predict the development of distant metastases and the presence of aggressive lung adenocarcinomas, respectively, in a statistically significant model. We conclude that serine hydrolase activities bear a predictive potential for human lung adenocarcinoma and that activity-based proteomics represents a powerful methodology in the search for novel disease biomarkers.

CXCL13 and CXCR5 expression by Non-Small Cell Lung Carcinoma (149.15)

Abstract Lung cancer is the leading cause of cancer deaths worldwide. Chemokines and their corresponding receptor have been shown to play important role in tumor progression, including lung cancer. In this regard, we have previously shown that the chemokine receptor CXCR5 is involved in metastatic dissemination in breast and prostate cancers. In this study we show CXCR5 is highly expressed by lung cancer cell lines and non-small cell lung carcinomas (NSCLC) tissues, than compared to non-neoplastic lung tissue. CXCL13, which is the only natural ligand for CXCR5, is also expressed within the lung tumors. Retrospective analysis of CXCR5 expression in 45 patients specimens showed significantly higher expression of CXCR5 in squamous cell carcinoma and adenocarcinoma samples. Interestingly, the nuclei of advanced tumor cells from these tissues were also positive for CXCR5 in more than 50% of these cases. Furthermore, ELISA showed significantly higher serum levels of CXCL13 by serum of patients with either adenocarcinoma or squamous cell carcinoma (n = 31), than compared to normal controls (n = 9). The mechanism underlying these clinically and biologically important findings need to be further explored.

Analysis of 2-[Fluorine-18]-Fluoro-2-deoxy-D-glucose Uptake Kinetics in PET Studies of Pulmonary Inflammation

Dynamic positron emission tomography (PET) imaging of the lung using the radiotracer 2-[fluorine-18]-fluoro-2-deoxy-D-glucose ((18)F-FDG) is an emerging method to assess noninvasively the metabolic activity of pulmonary inflammatory cells. Nevertheless, because of the distinct functional and structural characteristics of inflamed lung tissue standard methods of (18)F-FDG analysis can be substantially limited and there is no consensus about the best method for quantification of the (18)F-FDG signal for acute or chronic inflammatory lung diseases. This article gives an overview on recent advances in quantitative analysis of (18)F-FDG uptake kinetics in non-neoplastic inflamed lung tissue.

Relationship of aquaporin 1, 3, and 5 expression in lung cancer cells to cellular differentiation, invasive growth, and metastasis potential

An oncogenic capacity of aquaporins, transmembrane channels for water, was recently proposed. This study seeks to elucidate the involvement of aquaporin 1, 3, and 5 in the development and progression of lung cancer. Expression of aquaporin 1, 3, and 5 was examined by immunohistochemistry, Western blot, and laser-captured microdissection/real-time reverse transcription polymerase chain reaction in 160 lung cancers of various histologic subtypes; and its correlation with clinicopathological factors and survival was analyzed. Aquaporin 1, 3, and 5 were expressed in tumor cells in 71%, 40%, and 56% of lung cancers, respectively. Aquaporin expressions were frequent in adenocarcinomas, whereas aquaporin 1 and 5 were negative in squamous cell carcinomas. Bronchioloalveolar carcinoma cells exhibited an apicolateral aquaporin 1 and apicolateral or basolateral aquaporin 3 localization in nonmucinous type, and apical aquaporin 1 and 5 and basolateral aquaporin 3 expression in mucinous type, which corresponded to aquaporins expression of nonneoplastic lung tissue. Basolateral aquaporin 5 expression was acquired during tumorigenesis of nonmucinous bronchioloalveolar carcinoma. In contrast, invasive adenocarcinoma tumor cells overexpressed aquaporin 1 and 5 with loss of subcellular polarization and with an intracytoplasmic distribution. Overexpression of aquaporin 1 correlated with high postoperative adenocarcinoma metastasis ratios and unfavorable disease-free survival rates (P = .031). We conclude that expression patterns of aquaporin 1, 3, and 5 in lung cancer cells are mostly associated with cellular differentiation. However, the expression of aquaporin 1 and 5 is up-regulated in invading lung cancer cells, particularly in adenocarcinomas; and the overexpression of aquaporin 1 with loss of subcellular polarization is suggested to be involved in their invasive and metastatic potential.

On-the-spot lung cancer differential diagnosis by label-free, molecular vibrational imaging and knowledge-based classification

We report the development and application of a knowledge-based coherent anti-Stokes Raman scattering (CARS) microscopy system for label-free imaging, pattern recognition, and classification of cells and tissue structures for differentiating lung cancer from non-neoplastic lung tissues and identifying lung cancer subtypes. A total of 1014 CARS images were acquired from 92 fresh frozen lung tissue samples. The established pathological workup and diagnostic cellular were used as prior knowledge for establishment of a knowledge-based CARS system using a machine learning approach. This system functions to separate normal, non-neoplastic, and subtypes of lung cancer tissues based on extracted quantitative features describing fibrils and cell morphology. The knowledge-based CARS system showed the ability to distinguish lung cancer from normal and non-neoplastic lung tissue with 91% sensitivity and 92% specificity. Small cell carcinomas were distinguished from nonsmall cell carcinomas with 100% sensitivity and specificity. As an adjunct to submitting tissue samples to routine pathology, our novel system recognizes the patterns of fibril and cell morphology, enabling medical practitioners to perform differential diagnosis of lung lesions in mere minutes. The demonstration of the strategy is also a necessary step toward in vivo point-of-care diagnosis of precancerous and cancerous lung lesions with a fiber-based CARS microendoscope.

Downregulation of Signaling-active IGF-1 by Dipeptidyl Peptidase IV (DPP-IV)

Functioning as an extracellular protease, dipeptidyl peptidase IV (DPP-IV) preferentially cleaves the peptide bond after the penultimate proline residue. We report here that DPP-IV cleaves the first two amino acids from insulin-like growth factor 1 (IGF-1), revealed by mass spectrometry. The kinetic parameters of the proteolytic cleavage indicate that this reaction is physiologically relevant. Interestingly, truncated IGF-1 is less potent than the full-length protein in activating the IGF-1R, but binds more readily to IGF-binding protein 3 (IGFBP3). Quantitative RT-PCR showed that the level of DPP-IV mRNA is dramatically lower in lung squamous cell carcinoma tissues than in adjacent nonneoplastic lung tissues. However, this reduction was not observed in lung adenocarcinoma tissues. Our study suggests a possible link between IGF-1 and DPP-IV in cancer development in a specific tumor niche. A DPP-IV-related pathway may be important in mitigating IGF-1 signaling. Consequently, a robust IGF signaling pathway may accelerate early carcinogenesis in environments lacking DPP-IV.

ADAM28 is a serological and histochemical marker for non-small-cell lung cancers

ADAM28 (a disintegrin and metalloproteinase 28) is over-expressed in non-small-cell lung cancer (NSCLC) with correlation to cancer cell proliferation, tumor size and lymph node metastasis. The present study was aimed to develop an enzyme-linked immunosorbent assay (ELISA) system for diagnosis and monitoring of NSCLC. Our ELISA specifically measured ADAM28, showing negligible cross-reactivity with other metalloproteinases. The ADAM28 level in the NSCLC tissue was remarkably 36.9-fold higher than that in the non-neoplastic lung tissue (p < 0.001). The serum level was significantly 4.6-fold higher in the NSCLC patients (5.41 +/- 8.62 ng/ml; n = 102) than in the control subjects (1.17 +/- 0.93 ng/ml; n = 20) (p < 0.001), and increased with progress of tumor stage (p < 0.001). The level was also significantly higher in the patients with recurrent carcinoma than the control (p < 0.001) and in the patients with lymph node metastasis than those without metastasis (p < 0.001). The sensitivity, false-negative rate and AUC for ADAM28 were better than those for carcinoembryonic antigen. The combination of both assays improved the sensitivity, specificity, false-positive and false-negative rates for NSCLC. There was a positive correlation between the ADAM28 level measured by ELISA system and the degree of immunostaining in the lung adenocarcinomas with a size of <or=20 mm in diameter. The adenocarcinoma patients showing the high immunohistochemical reaction exhibited a poorer disease-free survival than those with the lower immunoreactivity (n = 102; p < 0.05). These data demonstrate that our ELISA is specific and sensitive to monitor the levels of ADAM28 in the samples from NSCLC patients and suggest that ADAM28 is a useful serological and histochemical marker for NSCLC.

Sarcoidosis development during induction chemotherapy for lung cancer mimicked progressive disease

We report a rare case of sarcoidosis that developed during induction chemotherapy for primary lung cancer, mimicking progressive disease. A 63-year-old man had an abnormal shadow in the right upper lung, and a bronchoscopic examination revealed a squamous cell carcinoma. Swelling of a pretracheal lymph node was also noted. Thus, we gave induction chemotherapy consisting of paclitaxel (days 1, 8) + carboplatin (days 1, 8) for two cycles under clinical staging of T2N2M0. After induction chemotherapy, (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) showed positive accumulation of FDG in mediastinal and bilateral hilar lymph nodes that had been negative in a previous FDG-PET examination, which led us to suspect disease progression. Transbronchial lymph node biopsy results showed sarcoid granulomas in the specimens. Following complete resection of the lung cancer, sarcoid granulomas were revealed in both nonneoplastic lung tissue and lymph nodes, which resulted in a diagnosis of lung cancer accompanied with sarcoidosis.

Metastasis-Associated Protein 1 Nuclear Expression is Associated with Tumor Progression and Clinical Outcome in Patients with Non-small Cell Lung Cancer

Little research has been done to test the usefulness of metastasis-associated protein 1 (MTA1) as a prognostic marker for non-small cell lung cancer (NSCLC). In this study, we investigated MTA1 expression and its prognostic value for NSCLC. NSCLC surgical tissue samples were taken from 100 patients with NSCLC who had been followed up for more than 2 years. The expression of MTA1 protein was evaluated by immunohistochemistry, and the correlations between the expression of MTA1 and clinical features and the prognosis were analyzed. The difference of MTA1 protein expression between NSCLCs and their adjacent nonneoplastic lung tissues was analyzed in a tissue microarray. The change of MTA1 mRNA expression and protein expression after RNA interference (RNAi) was detected by real-time polymerase chain reaction, Western blot, and immunocytochemistry in NSCLC cell line 95D. Overexpression of MTA1 (immunoreactivity scoring >4) was shown in 61.0% of the NSCLC cases but only in 9.4% of their adjacent nonneoplastic lung tissues (p < 0.001). The MTA1 expression level was correlated with lymph node metastasis (p = 0.013) and clinical stage (p = 0.002). Survival analysis showed that the MTA1 overexpression group had a significantly shorter overall survival time than the MTA1 downexpression group (p = 0.003). However, multivariate analysis showed that MTA1 expression was not a significant and independent prognostic parameter for patients with NSCLC. After RNAi, the 95D cells exhibit consistent reduction in MTA1 mRNA expression and MTA1 protein expression. MTA1 protein expression is associated with tumor progression and clinical outcome in patients with NSCLC. Further molecular, cellular, and animal model studies on MTA1 gene will provide new clues for exploring the mechanism of carcinogenesis and tumor progression in patients with NSCLC.

Evaluation of ADAM28 as a serological and histochemical marker for non-small cell lung cancers.

10599 Background: ADAMs (a disintegrin and metalloproteinases) are a gene family, which exhibit both proteolytic and adhesive properties. ADAM28 is over-expressed in non-small cell lung cancer (NSCLC) with correlation to cancer cell proliferation, tumor size and lymph node metastasis. This study was aimed to develop an enzyme-linked immunosorbent assay (ELISA) system for serological and histochemical marker of NSCLC. Methods: AnELISA system by using two monoclonal antibodies against ADAM28. Serum levels of ADAM28 in 102 NSCLC and 20 controls were measured. ADAM28 was also examined in urine samples. ADAM28 production levels in the resected specimens were compared with the degree of immunoreactivity. The patients were classified to three groups (high-, middle- and the low-expressing) according to the immunostaining and analyzed by the Kaplan-Meier. Results: Our ELISA specifically measured ADAM28, showing negligible cross-reactivity with other metalloproteinases. The ADAM28 level in the NSCLC tissue was remarkably 36.9-fold higher than that in the non-neoplastic lung tissue (p < 0.001). The serum level was significantly 4.6-fold higher in NSCLC patients (5.41 ± 8.62 ng/mL) than in controls (1.17 ± 0.93 ng/mL) (p < 0.001), and increased with progress of stage. The level was also significantly higher in the patients with recurrence than controls (p < 0.001) and in the patients with lymph node metastasis than those without metastasis (p < 0.001). The sensitivity, false-negative rate and AUC for ADAM28 were better than those for CEA. The detection rate in urine was increased with progress of stage, and significant correlated with serum subjects (n = 45; p < 0.001). There was a positive correlation between the ADAM28 level and the degree of immunostaining in the lung adenocarcinomas with a size of ≤ 20 mm in diameter. The patients showing the high immunohistochemical reaction exhibited a poorer disease-free survival (n = 102; p < 0.05). Conclusions: Our data demonstrate that our ELISA is specific and sensitive to monitor the levels of ADAM28 in NSCLC and suggest that ADAM28 is a useful serological and histochemical marker for NSCLC. No significant financial relationships to disclose.

EML4-ALK Rearrangement in Non-Small Cell Lung Cancer and Non-Tumor Lung Tissues

A fusion gene, echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK), with transforming activity has recently been identified in a subset of non-small cell lung cancer (NSCLC), but its pathogenetic, diagnostic, and therapeutic roles remain unclear. Both frequency and type of EML4-ALK transcripts were investigated by reverse transcription PCR in 120 frozen NSCLC specimens from Italy and Spain; non-neoplastic lung tissues taken far from the tumor were used as controls. In cases carrying the fusion transcript, we determined EML4-ALK gene and protein levels using fluorescence in situ hybridization, Western blotting, and immunoprecipitation. We also analyzed ALK protein levels in paraffin samples from 662 NSCLC specimens, including the 120 cases investigated in the molecular studies. EML4-ALK transcripts (variants 1 and 3) were detected in 9 of 120 NSCLC samples but were not specific for NSCLC since they were also found in non-cancerous lung tissues taken far from the tumor. Notably, no transcripts were detected in matching tumor samples from these patients. Fluorescence in situ hybridization analysis of cases expressing EML4-ALK transcripts showed that only a minority of cells harbored the EML4-ALK gene. None of these cases was found to express the EML4-ALK protein as examined by immunohistochemistry, Western blotting, and immunoprecipitation. The EML4-ALK transcript cannot be regarded as a specific diagnostic tool for NSCLC. Our results show therefore that the causal role and value of EML4-ALK as a therapeutic target remain to be defined.

The redox state of the lung cancer microenvironment depends on the levels of thioredoxin expressed by tumor cells and affects tumor progression and response to prooxidants

Here we report that human nonsmall cell lung carcinomas overexpress macrophage migration inhibitory factor (MIF) and thioredoxin (Trx), 2 oxidoreductases with cytokine function, and contain more abundant nonprotein thiols (glutathione and cysteine) than nonneoplastic lung tissues. Cell clones derived from the same lung carcinoma cell lines but expressing different levels of Trx and/or MIF displayed growth rates in vitro and in vivo correlating with Trx but not with MIF. Interestingly, the different clones generate extracellularly reduced nonprotein thiols, in amounts related to the Trx content and inhibited by inhibitors of Trx function. Each clone also showed distinct responses to the prooxidant compound arsenic trioxide. Cells with a strongly antioxidant and aggressive phenotype were more susceptible to the cytotoxic effect of the drug than cells expressing little Trx. The latter counteracted the oxidative stress by increasing Trx expression and thiol release. Together these results indicate that different human lung cancer cell lines have distinct redox properties defined by the levels of Trx and nonprotein thiols, the higher antioxidant phenotype correlating with the higher aggressiveness. Moreover, the redox phenotype dictates their response to prooxidant drugs and must be taken into account when therapeutic interventions with redox active substances are considered.

Intratumoral Estrogens and Estrogen Receptors in Human Non–Small Cell Lung Carcinoma

The possible involvement of gender-dependent factors has been suggested in human non-small cell lung carcinomas (NSCLC), but their precise roles remain largely unclear. Therefore, we examined intratumoral estradiol concentrations in NSCLC to examine local actions of estrogens in NSCLC. Fifty-nine frozen specimens of NSCLC were available for liquid chromatography/electrospray tandem mass spectrometry to study intratumoral estradiol concentrations. In addition, A549 NSCLC cells stably expressing estrogen receptor (ER) alpha (A549 + ERalpha) or ERbeta (A549 + ERbeta) were used in vitro studies. Forty-three (73%) of 59 NSCLC showed higher concentration of estradiol in carcinoma tissues than the corresponding nonneoplastic lung tissues from the same patient, and intratumoral estradiol concentrations were significantly (P = 0.0002 and 2.2-fold) higher than the corresponding nonneoplastic lungs. The intratumoral concentration of estradiol was positively correlated with aromatase expression, tumor size, and Ki-67 status in ERalpha- or ERbeta-positive cases. In in vitro studies, estradiol significantly increased cell proliferation of A549 + ERalpha or A549 + ERbeta, which was significantly suppressed by selective ER modulators, tamoxifen or raloxifene. Both A549 + ERalpha and A549 + ERbeta cells expressed aromatase. The cell proliferation level in these cells was significantly increased under treatment with testosterone, and it was inhibited by addition of the aromatase inhibitor letrozole. These results suggest that estradiol is locally produced in NSCLC mainly by aromatase and plays an important role in the growth of ERalpha- or ERbeta-positive NSCLC. Therefore, use of selective ER modulators and/or aromatase inhibitors may be clinically effective in NSCLC that are positive for both ER and aromatase.

Significance of the expression of Rho-GDP dissociation inhibitorβ,γ in lung squamous cell carcinoma and adenocarcinoma

ObjectiveTo study the expression of Rho-GDP dissociation inhibitor β, γ (Rho-GDIβ, Rho-GDIγ) in lung squamous cell carcinoma and adenocarcinoma and its relationship with the expression of RhoC (Ras homologus oncogenes C) and clinicopathologic parameters.MethodsWestern blot assay was employed for Rho-GDIβ, Rho-GDIγ and RhoC in lung squamous cell carcinoma and adenocarcinoma and non-neoplastic lung tissues of 37 cases with fresh specimens.ResultsThe study showed that Rho-GDIβ, Rho-GDIγ and RhoC were expressed in lung cancer and non-neoplastic lung tissues, the level in lung cancer tissue was much higher than that in non-neoplastic tissues (P<0.001). In lung cancer, the expression of Rho-GDIβ was much higher in patients with lymph node metastasis (P=0.021), and the expression of Rho-GDIγ was much higher in poorly differentiated tumor than in well-differentiated and moderately differentiated tumor, but both of them were not correlated with other clinicopathologic parameters. The expressions of Rho-GDIβ and Rho-GDIγ were not correlated with the expression of RhoC.ConclusionIn lung cancer, Rho-GDIβ and Rho-GDIγ may play a role in the tumorigenesis, Rho-GDIβ may promote metastasis, and Rho-GDIγ may have some relationship with differentiation.

Aberrant methylation of E‐cadherin and H‐cadherin genes in nonsmall cell lung cancer and its relation to clinicopathologic features

Lung cancer is the leading cause of cancer deaths worldwide. The epigenetic inactivation of certain genes by aberrant promoter methylation plays an important role in the pathogenesis of lung cancer. In addition, DNA hypermethylation is an extremely promising cancer marker. Cadherins are cell adhesion molecules that modulate the epithelial phenotype and regulate tumor invasion. Although the aberrant methylation of E-cadherin (CDH1) or H-cadherin (CDH13) genes has been reported in lung cancer, to the authors' knowledge, the relation between the concurrent hypermethylation in E-cadherin and H-cadherin has not been explored to date. The authors investigated the methylation status of the promoter region of human E-cadherin and H-cadherin genes in resected samples of primary nonsmall cell lung cancer (NSCLC) using methylation-specific polymerase chain reaction (MSP) analysis and correlated the results with clinicopathologic characteristics. The methylation status of the promoter regions of the E-cadherin and H-cadherin genes was examined by using nested MSP in 88 primary lung cancers and paired adjacent normal tissues. Data were compared with clinicopathologic features. The frequency of methylation in neoplastic and corresponding nonneoplastic lung tissues was 30 of 88 tissue samples (34.1%) and 5 of 88 tissue samples (5.7%) for E-cadherin and 26 of 88 tissue samples (29.5%) and 7 of 88 tissue samples (8%) for H-cadherin, respectively. In addition, in 9 patients (10.2%), both genes were methylated. Although there was no significant difference in the overall survival of patients according to the methylation pattern of the E-cadherin gene alone or the H-cadherin gene alone, patients with NSCLC who had hypermethylation of both genes had a significantly longer overall survival. However, no correlation was observed between their methylation status and any other clinicopathologic factors. The current findings suggested that simultaneous methylation of the E-cadherin and H-cadherin genes occurs in a subset of NSCLCs and may be used as a prognostic marker for patients with NSCLC. However, further studies with large numbers of patients will be needed to confirm the findings.