Non-small Cell Lung Cancer Tissue Samples Research Articles

Comparison of the expression levels of napsin A, thyroid transcription factor-1, and p63 in nonsmall cell lung cancer using cytocentrifuged bronchial brushings

Appropriate treatment of nonsmall cell lung cancer (NSCLC) depended on histologic type. The aim of this study was to evaluate the expression levels of Napsin A, thyroid transcription factor-1 (TTF-1), and p63 by immunostaining using CytoRich Red preserved cytocentrifuged bronchial brushings. The expression levels of Napsin A, TTF-1, and p63 were semiquantitatively evaluated using proportion and intensity scores in 12 patients from whom both bronchial brushing cytology and bronchial biopsy samples had been obtained, as well as in a selection of 64 resected NSCLC tissue samples, and 8 normal or benign bronchial brushing cytology samples. The nuclear expressions of TTF-1 and p63 allowed for good visualization of cancer cells, whereas the cytoplasmic expression of Napsin A in cancer cells was often more difficult to evaluate because medium-sized and large macrophages also expressed Napsin A. Napsin A and TTF-1 were reliable adenocarcinoma markers, and p63 was a reliable squamous cell carcinoma marker in cytology, and histology samples. The average scores of Napsin A, TTF-1, and p63 were highly correlated in bronchial brushing cytology and bronchial biopsy samples, whereas TTF-1 expression was significantly lower than Napsin A expression in resected NSCLC tissue samples (P < .001). Immunostaining of bronchial brushings fixed with CytoRich Red was useful in determining histologic types in NSCLC. It was suggested that the panels of Napsin A, TTF-1, and p63 were effective in identifying histologic type in NSCLC, and that a combination of either Napsin A and p63 or TTF-1 and p63 should be chosen, depending on morphology.

DJ-1 may contribute to metastasis of non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients' tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (P = 0.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (P = 0.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (P = 0.039). DJ-1 might contribute to the metastasis of NSCLC.

Prognostic significance of STAT3 expression and its correlation with chemoresistance of non-small cell lung cancer cells

Activation of signal transducer and activator of transcription 3 (STAT3) plays important roles in tumorigenesis and tumor development. Previously, we have reported that overexpression of STAT3 potentiates growth, survival and radioresistance of non-small cell lung cancer (NSCLC) cells. The aim of this study was to investigate the prognostic significance of STAT3 expression and its correlation with chemoresistance of NSCLC cells. Semi-quantitative RT-PCR was performed to detect the expression of STAT3 mRNA in 12 NSCLC and corresponding adjacent lung tissues. Immunohistochemistry was performed to detect the expression of STAT3 protein in 76 NSCLC tissue samples. Additionally, the correlation between STAT3 expression and prognosis of NSCLC patients was statistically analyzed. The role of STAT3 in chemoresistance of NSCLC cells was also assessed by the vector-based small interfering RNA. The expression level of STAT3 mRNA in NSCLC tissues was significantly higher than that in corresponding adjacent lung tissues (P<0.05). Positive immunostaining of STAT3 protein was mainly located in the cytoplasm of tumor cells. The expression of STAT3 protein was significantly correlated with tumor differentiation, clinical stage and lymph node metastasis of NSCLC patients. Moreover, the 5-year overall survival rate of patients with high STAT3 expression (42.3%) was significantly lower than that of patients with low STAT3 expression (58.8%; P<0.001). Multivariate analysis using the Cox proportional hazard model showed that high STAT3 protein expression was an independent prognostic factor for NSCLC patients (P=0.021). Furthermore, two stably transfected cell lines (A549/shSTAT3 and SPC-A1/shSTAT3) were successfully established, and RNAi-mediated STAT3 inhibition could significantly increase the sensitivity of NSCLC cells to cisplatin by enhancing caspase-3-dependent apoptosis. Together, the expression of STAT3 might be an independent prognostic marker for NSCLC patients and RNAi-mediated STAT3 inhibition would be a potential strategy for chemosensitization of NSCLC cells.

C057 Speech performance and sound localization abilities in Neurelec Digisonic(r) SP Binaural cochlear implant users

Although numerous dysregulated circular RNAs (circRNAs) was discovered, the study between circRNAs and non-small cell lung cancer (NSCLC) is just beginning. Here, we investigated the functions and provided a possible mechanism of circ_0000735 for NSCLC. Real-time PCR was used to elucidate the level of circ_0000735 in NSCLC tissue samples and cells. CCK8, colony formation, flow cytometric, and transwell experiments were carried out to evaluate cell proliferation, apoptosis, migratory and invasive abilities in NSCLC cell lines. Luciferase reporter assays were conducted to elucidate the mechanisms of circ_0000735 in NSCLC. Circ_0000735 was elevated in both NSCLC tissues and cells. This upregulation of circ_0000735 is associated with more advanced TNM stages and lymph node invasion. Gain and loss of function experiments documented that circ_0000735 significantly facilitated cell proliferation, migratory and invasive abilities and inhibit cell apoptosis. Moreover, we proved circ_0000735 could bound to miR-1179/1182 to exert its biological functions in NSCLC cells. Taken together, this work provided a possible treatment target for this devastating disease.

Upregulation of microRNA-451 increases cisplatin sensitivity of non-small cell lung cancer cell line (A549)

BackgroundRecently, miR-451 as a tumor suppressor has been reported in other studies. However, whether miR-451 can affect the sensitivity of non-small cell lung cancer (NSCLC) cells to cisplatin (DDP) remains unclear. The aim of this study is to evaluate the roles of miR-451 in the sensitivity of NSCLC cells to DDP.MethodsQuantitative RT-PCR assay was performed to detect the expression of miR-451 in 10 pairs of NSCLC and noncancerous tissue samples. pcDNA-GW/EmGFP-miR-451 was stably transfected into NSCLC cell line (A549). Then, the effects of miR-451 upregulation on growth, colony formation and apoptosis of A549 cells were investigated. Finally, the effects of miR-451 upregulation on in vitro and in vivo sensitivity of A549 cells of DDP were also determined.ResultsThe level of miR-451 expression in NSCLC tissues was significantly higher than that in corresponding noncancerous tissues. Ectopic overexpression of miR-451 could significantly inhibit growth and induce apoptosis of A549 cells. Moreover, ectopic overexpression of miR-451 could sensitize A549 cells to DDP possibly by increasing DDP-induced apoptosis which might be associated with the inactivation of Akt signaling pathway.ConclusionsThis study demonstrated for the first time that combination of DDP application with miR-451 upregulation might be a potential strategy for the treatment of human NSCLC.

Personalizing treatment with pemetrexed (PEM) in patients with malignant mesothelioma (MPM) and non-small cell lung cancer (NSCLC): The role of thimidylate synthase.

e21001 Background: PEM was first approved for the treatment of MPM in combination with cisplatin, more recently as first line and maintenance therapy for advanced nonsquamous NSCLC. TS levels may predict response to PEM; high levels of expression are speculated to predict resistance to PEM. To evaluate whether TS protein levels, rather than histologic type, could be used to select patients with NSCLC and MPM for treatment with PEM, we assessed the variability of expression of TS in NSCLC and MPM tissue samples. Methods: We pursued semi-quantification of TS protein levels by immunohistochemistry (IHC) on tissue microarrays from 83 adenocarcinoma, 92 squamous, large cell, not-other-specified (NOS) NSCLC and 40 MPM samples. An IHC scoring system (IRS) was implemented based on stains: IRS scores of 0-2, 3-7 and 8-12 signified low, moderate and high TS protein expression respectively. Results: Of 49 squamous-cell samples, 22% had low expression of TS, indicating that PEM could potentially be effective in a subset of patients with squamous lung cancer. 24% expressed high levels of TS, 55% had an IRS greater than 6, in contrast to 23% of the adenocarcinoma samples. Of 83 adenocarcinoma samples, 56.5% and 9.6% had moderate and high expression of TS respectively. Similarly, 47% and 35% of the MPM samples showed moderate and high levels of TS expression respectively (Table). Correlation of clinical response to PEM with levels of TS expression is ongoing. Conclusions: Based on these preliminary data, TS levels are highly variable across both NSCLC and MPM tissue samples, and a substantial minority of patients with squamous histology may have a molecular profile that correlates with sensitivity to PEM. The role of TS as a biomarker for personalized therapy with PEM in patients with NSCLC and MPM requires prospective validation. TS expression Tumor type Negative (IRS = 0-2) Low (IRS = 3-7) High (IRS = 8-12) Mesothelioma (n = 40) 17.5% 47.5% 35% Adeno (n = 83) 33.6% 56.5% 9.6% Squamous, large cell, and NOS (n=92) 23.8% 50.8% 25% Squamous (n = 49) 22.2% 50.8% 24.4% Large cell (n = 11) 18% 54% 27.2% NOS/BAC (n = 12) 16.6% 41.5% 41.6% No significant financial relationships to disclose.

High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

Background:Carbonic anhydrase IX (CAIX) is an enzyme upregulated by hypoxia during tumour development and progression. This study was conducted to assess if the expression of CAIX in tumour tissue and/or plasma can be a prognostic factor in patients with non-small cell lung cancer (NSCLC).Methods:Tissue microarrays containing 555 NSCLC tissue samples were generated for quantification of CAIX expression. The plasma level of CAIX was determined by ELISA in 209 of these NSCLC patients and in 58 healthy individuals. The CAIX tissue immunostaining and plasma levels were correlated with clinicopathological factors and patient outcome.Results:CAIX tissue overexpression correlated with shorter overall survival (OS) (P=0.05) and disease-specific survival (DSS) of patients (P=0.002). The CAIX plasma level was significantly higher in patients with NSCLC than in healthy individuals (P<0.001). A high level of CAIX in the plasma of patients was associated with shorter OS (P<0.001) and DSS (P<0.001), mostly in early stage I+II NSCLC. Multivariate Cox analyses revealed that high CAIX tissue expression (P=0.002) was a factor of poor prognosis in patients with resectable NSCLC. In addition, a high CAIX plasma level was an independent variable predicting poor OS (P<0.001) in patients with NSCLC.Conclusion:High expression of CAIX in tumour tissue is a predictor of worse survival, and a high CAIX plasma level is an independent prognostic biomarker in patients with NSCLC, in particular in early-stage I+II carcinomas.

Biological significance of the maximum standardized uptake values on positron emission tomography in non‐small cell lung cancer

The 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) has recently become an important non-invasive tool for the diagnosis and staging in several cancers. The standardized uptake value (SUV) of primary tumor has been reported to relate to cancer progression and prognosis, however, biological mechanism is still unclear. Seventy-nine patients with non-small cell lung cancer (NSCLC) who had undergone preoperative FDG-PET and a surgical resection were enrolled in this study. NSCLC tissue samples prepared from the surgical specimens were subjected to an immunohistochemical analysis for the expression of Ki-67 and vascular endothelial growth factor (VEGF) proteins. The relationships between the expression status of these proteins and SUV(max) of primary tumors were evaluated. Concerning the relationship with various clinicopathological findings, SUV(max) of primary tumors was associated with histology, tumor proliferation, pleural or vascular invasion, and pathological stage. A significant correlation was observed between the SUV(max) and either the Ki-67 or VEGF expression (P < 0.001, P = 0.006), respectively. Cases with both Ki-67-negative and VEGF-negative findings exhibited a significantly lower SUV(max) than those with single positive or double positive cases (P = 0.006, P < 0.001). The SUV(max) was associated with the expression of Ki-67 and VEGF in NSCLC. These findings indicated that the SUV(max) of primary tumors might therefore reflect the biological malignant potential in NSCLC.

Prognostic significance of VEGF-C expression in correlation with COX-2, lymphatic microvessel density, and clinicopathologic characteristics in human non-small cell lung cancer

Lung cancer is one of the most lethal cancers in China because of high incidence and high mortality. Cyclooxygenase-2 (COX-2) and vessel endothelial growth factor C (VEGF-C) were found to play an important role in lymphangiogenesis of malignant tumors. In this study, we investigated whether lymphatic microvessel density (LMVD) is related to the prognosis in non-small cell lung cancer (NSCLC) patients, and the expressions of COX-2 and VEGF-C so as to determine the possible role of COX-2 and VEGF-C in NSCLC lymphangiogenesis. Sixty-five formalin-fixed paraffin embedded tissue samples of NSCLC were evaluated for COX-2 and VEGF-C by immunohistochemical staining. To assess tumor lymphangiogenesis, LMVD was determined by immunohistochemical staining of VEGFR-3 polyclonal antibody. The relationship among COX-2 and VEGF-C expression, LMVD, and clinicopathologic parameters was analyzed. Among the 65 samples, high LMVD was significantly associated with lymph node metastasis and poor survival. Multivariate survival analysis showed that LMVD value and lymph node metastasis were independent prognostic factors. The expression level of COX-2 and VEGF-C was significantly higher than those of the adjacent tissues. COX-2 and VEGF-C expressions in NSCLC significantly correlated with lymph node metastasis, but not with patient gender, age, tumor size, or tumor, nodes, metastasis classification stage. The mean LMVD value of COX-2- or VEGF-C-positive tumors was higher than that of COX-2- or VEGF-C-negative tumors. A significant correlation was found between the expressions of COX-2 and VEGF-C. This study suggests that LMVD may be one of the important prognostic factors for NSCLC patients. VEGF-C might play an important role in the COX-2 lymphangiogenic pathway. COX-2 and VEGF-C may play an important role in tumor progression by stimulating lymphangiogenesis. The inhibition of lymphangiogenesis, COX-2, or VEGF-C activity may have an important therapeutic benefit in the control of NSCLC.

The Expression of Three Genes in Primary Non–Small Cell Lung Cancer Is Associated with Metastatic Spread to the Brain

Brain metastases affect 25% of patients with non-small cell lung cancer (NSCLC). We hypothesized that the expression of genes in primary NSCLC tumors could predict brain metastasis and be used for identification of high-risk patients, who may benefit from prophylactic therapy. The expression of 12 genes was measured by real-time quantitative reverse transcriptase PCR in 142 frozen NSCLC tissue samples. Univariate and multivariate Cox regression analysis was used to analyze the correlation between gene expression and the occurrence of brain metastasis. Immunohistochemistry on independent samples was used to verify the findings. A score based on the expression levels of three genes, CDH2 (N-cadherin), KIFC1, and FALZ, was highly predictive of brain metastasis in early and advanced lung cancer. The probability of remaining brain metastasis-free at 2 years after diagnosis was 90.0+/-9.5% for patients with stage I/stage II tumors and low score compared with 62.7+/-12% for patients with high score (P<0.01). In patients with more advanced lung cancer, the brain metastasis-free survival at 24 months was 89% for patients with low score compared with only 37% in patients with high score (P<0.02). These results were confirmed by immunohistochemical detection of N-cadherin in independent cohort of primary NSCLC. The expression levels of three genes in primary NSCLC tumors may be used to identify patients at high risk for brain metastasis who may benefit from prophylactic therapy to the central nervous system.

Interference With Netrin-1 and Tumor Cell Death in Non–Small Cell Lung Cancer

Netrin-1 may promote colorectal and breast tumorigenesis, by inhibiting apoptosis induced by its dependence receptors, deleted in colorectal cancer (DCC) and uncoordinated-5-homolog (UNC5H). The status of netrin-1 and its receptors in non-small cell lung cancer (NSCLC) was unknown. The levels of netrin-1 and its receptors were analyzed in a panel of 92 NSCLC and 25 human lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. In lung cancer cell lines that express netrin-1, the expression of netrin-1 was inhibited by using small interfering RNA (siRNA), or interference with netrin-1 was performed by treatment with a decoy recombinant DCC ectodomain protein (DCC-5Fbn). Cell death was monitored with a trypan blue exclusion assay or by measuring caspase-3 activity. The effect of netrin-1 interference on tumor growth was analyzed by DCC-5Fbn intratumoral or netrin-1 siRNA intraperitoneal injection in mice engrafted with lung cancer cell lines. All statistical tests were two-sided. High levels of netrin-1 were found in 43 of the 92 NSCLC tumor samples (47%). Interference with netrin-1 in human lung cancer cell lines was associated with UNC5H-mediated cell death in vitro (percentage of cell death in untreated and in DCC-5Fbn-treated cells = 8% and 26%, respectively, difference = 18%, 95% confidence interval [CI] = 10% to 26%; P = .049) and with lung tumor growth inhibition and/or regression in xenografted nude mice (12 mice in DCC-5Fbn-treated group and 13 mice in control group). Mean volume of control and DCC-5Fbn-treated tumors on day 46 was 489 and 84 mm(3), respectively (difference = 404 mm(3), 95% CI = 145 to 664 mm(3); P < .001). Almost half of the NSCLC tissue samples examined expressed high levels of netrin-1. Extracellular targeting of the interaction between netrin-1 and UNC5H may be a promising therapeutic approach for NSCLCs that express netrin-1.

Targeting Dishevelled, a mediator of Wnt/β-Catenin Signaling Pathway, in Human Non—Small-Cell Lung Cancer

Lung cancer is the leading cause among cancer-related deaths worldwide. The Wnt/β-catenin signaling pathway has been proven to be activated in non—small-cell lung cancer (NSCLC). We have previously reported that Dishevelled-3 (Dvl-3), interacting with the intracellular domain of Wnt signal receptor Frizzled (Fz) and functioning as a key mediator of the Wnt/β-catenin pathway, is over-expressed in NSCLC tissue samples. We also found that knocking down Dvl-3 by siRNA suppressed NSCLC cell growth through downregulating cytosolic β-catenin level and TCF-dependent transcriptional activity, hallmarks of activation of the Wnt/β-catenin signaling pathway. Moreover, knocking down Dvl-3 induced cell-cycle arrest and sensitized NSCLC cells to cisplatin treatment. More recently, we rationally designed a small-molecule inhibitor that interrupted the interaction between the intracellular domain of Fz receptor and the PDZ domain of Dvl. We found that this compound downregulated the Wnt/β-catenin signaling pathway induced apoptosis in NSCLC cells in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that small-molecular inhibition of the oncogenic Dvl function might have potential therapeutic implications in the treatment of patients with NSCLC.

Expressions of Livin and Smac proteins in non-small cell lung cancer

To investigate the expression characteristics of Livin and second mitochondrial activator of Caspase (Smac) proteins in non-small cell lung cancer (NSCLC), and analyze their effect on patients’ prognosis. The expressions of Livin and Smac proteins were detected in 89 NSCLC tissue samples and 25 normal lung tissue samples by immunohistochemical technique. The positive expression rates of Livin and Smac proteins in NSCLC tissues were 53.9%, and 58.4% respectively, higher than that in normal lung tissues (P<0.01). Livin protein expression correlated with Smac protein significantly (x 2=18.451, P=0.000, r=0.455). The expression level of Livin protein was closely related to lymph node metastasis, TNM stage and histological type (P<0.05), but not to sex, age, differentiation grade (P>0.05). The expression level of Smac protein was closely related to lymph node metastasis, TNM stage (P<0.01), but not to sex, age, histological types (P>0.05). Kaplan-Meier analysis revealed a significant impact on survival by Livin protein in NSCLC (P<0.01), but not by Smac protein (P>0.05). Overexpression of Livin protein may play a promoting role in the occurrence and progression of NSCLC. Moreover, it may bring an adverse effect on patients’ prognosis. Although overexpression of Smac protein affects the occurrence and progression of NSCLC, it has no relationship with the prognosis. Livin protein may be helpful to evaluate the progression of NSCLC, and to predict the prognosis.

Epidermal growth factor receptor (EGFR) gene copy number detection in non‐small‐cell lung cancer; a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization

The epidermal growth factor receptor (EGFR) is an important target for anticancer therapy. In non-small-cell lung cancer (NSCLC), mutations in the tyrosine kinase domain of EGFR and EGFR gene copy number have been demonstrated to identify patients most likely to benefit from EGFR tyrosine kinase inhibitors. EGFR gene copy number has been assessed mainly by fluorescence in situ hybridization (FISH), a method requiring the use of a fluorescence microscope and often hampered by the rapid fading of the fluorescent signal. These limitations of FISH can be overcome by using chromogenic in situ hybridization (CISH). To test the applicability of CISH for EGFR gene copy number testing in NSCLC, a comparison of CISH and FISH was performed. A total of 58 formalin-fixed, frozen NSCLC tissue samples were collected on which both CISH and FISH were performed. High concordance was found in the assessment of EGFR copy number between observers and between techniques (kappa coefficient = 0.64-0.76). CISH seemed the ideal technique for paraffin sections, whereas FISH was favourable for frozen material. CISH is a suitable alternative strategy to FISH in determining EGFR gene copy number in NSCLC patients.

Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

We developed and validated a real-time reverse transcription (RT)-PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (alpha+beta+, alpha-beta+, alpha+beta-, alpha-beta-) in tumor cell lines and non-small cell lung cancer (NSCLC). We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript-specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The alpha+beta- splice variant showed the highest expression and alpha-beta+ and alpha-beta- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the alpha+beta- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by alpha+beta+, with the alpha-beta+ and alpha-beta- splice variants having the lowest expression. In the NSCLC tumors, the alpha+beta+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT alpha+beta+ splice variant may be an independent negative prognostic factor for NSCLC patients.

MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the most frequent and therapy-refractive sub-class of lung cancer. Improving apoptosis induction in NSCLC represents a logical way forward in treating this tumor. Cisplatin, a commonly used therapeutic agent in NSCLC, induces activation of N-terminal-c-Jun kinase (JNK) that, in turn, mediates induction of apoptosis. In analysing surgical tissue samples of NSCLC, we found that expression of MKP1/CL100, a negative regulator of JNK, showed a strong nuclear staining for tumor cells, whereas, in normal bronchial epithelia, MKP1 was localized in the cytoplasm as well as in nuclei. In the NSCLC-derived cell lines H-460 and H-23, we found that MKP1 was constitutively expressed. Expressing a small-interfering RNA (siRNA) vector for MKP1 in H-460 cells resulted in a more efficient activation by cisplatin of JNK and p38 than in the parental cells, and this correlated with a 10-fold increase in sensitivity to cisplatin. A similar response was also observed in H-460 and H-23 cells when treated with the MKP1 expression inhibitor RO-31-8220. Moreover, expression of a siRNA-MKP2, an MKP1-related phosphatase, had no effect on H-460 cell viability response to cisplatin. Tumors induced by H-460 cells expressing MKP1 siRNA grew slower in nu(-)/nu(-) mice and showed more susceptibility to cisplatin than parental cells, and resulted in an impaired growth of the tumor in mice. On the other hand, overexpression of MKP1 in the H-1299 NSCLC-derived cell line resulted in further resistance to cisplatin. Overall, the results showed that inhibition of MKP1 expression contributes to a slow down in cell growth in mice and an increase of cisplatin-induced cell death in NSCLC. As such, MKP1 can be an attractive target in sensitizing cells to cisplatin to increase the effectiveness of the drug in treating NSCLC.

P16INK4a Promoter Hypermethylation in Sputum, Blood, and Tissue from Non-Small Cell Lung Cancer and Pulmonary Inflammation

Background : The aberrant promoter hypermethylation of p16INK4a, as a tumor suppressor gene, is contributory factor to non-small cell lung cancer(NSCLC). However, its potential diagnostic impact of lung cancer is unclear. This study measured the level of promoter hypermethylation in the sputum and blood, and compared this with the level measured in the tissue obtained from NSCLC and pulmonary inflammation. Methods : Of the patients who visited the Our Lady of Mercy Hospital in Incheon, Korea for an evaluation of a lung mass and underwent blood, sputum, and tissue tests, 23patients (18 NSCLC, 5 pulmonary inflammation) were enrolled in this study. DNA was extracted from each sample and the level of p16INK4amethylation was determined using methylation-specific polymerase chain reaction. Results : methylation of the blood was observed in 88.9% (16 of 18) and 20.0% (1 of 5) of NSCLC and from pulmonary inflammation samples, respectively (P=0.008). Methylation of the sputum was observed in 83.3% (10 of 12) 80.0% (4 of 5) of NSCLC and pulmonary inflammation samples, respectively (P=1.00). Among the 8 NSCLC tissue samples, methylation changes were detected in 75.0% of samples (6 cases). Four out of seven tissue samples (57.1%) showed concordance, being methylated in both the blood and sputum. Conclusions : There was a higher level of methylation of the blood from NSCLC patients than from pulmonary inflammation. The tissue showed a high concordance with the blood in the NSCLC samples. These findings suggest that promoter hypermethylation of the blood can used to discriminate between NSCLC and pulmonary inflammation.

Immunocytochemical detection of deoxycytidine kinase in haematological malignancies and solid tumours

Background: Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important deoxynucleoside analogues used for the treatment of haematological and solid malignancies. Aim: To measure dCK expression in...

Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas

Both epidermal growth factor receptor (EGFR) and RAS gene mutations contribute to the development of non-small cell lung cancer (NSCLC). Because RAS is one of the downstream molecules in the EGFR signal transduction, the association between the somatic mutations of EGFR and RAS may be important in the pathogenesis of NSCLC . However, to date, such data are lacking. In this study, we analyzed the hotspot regions of K-RAS gene (codons 12, 13, 59 and 61) and EGFR gene (exons 18, 19 and 21) in 153 NSCLC tissue samples including 69 adenocarcinomas. Overall, we detected 30 EGFR mutations (19.6%) and 6 K-RAS mutations (3.9%) in the 153 NSCLCs. In the 69 adenocarcinomas, 26 EGFR mutations (37.7%) and six K-RAS mutations (8.7%) were detected. Of note, the 26 tumors with EGFR mutations did not harbor any K-RAS mutations, and the six tumors with K-RAS mutations did not harbor any EGFR mutations. Inverse relationship between K-RAS and EGFR mutations in the lung adenocarcinoma was statistically significant (P=0.046, chi2 test). As regards smoking history, EGFR mutation was significantly associated with never-smoking history, whereas K-RAS mutation was significantly associated with smoking history. Our data suggest that mutations of EGFR and K-RAS genes might separately, but not cooperatively, contribute to lung adenocarcinoma pathogenesis, and that EGFR and K-RAS mutants could separately be anti-neoplastic targets in lung adenocarcinomas.