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Gemcitabine is a promising compound for the treatment of human lung cancer. Although apoptosis has been shown to play a role in certain cell types with gemcitabine, the steps leading to cell death after the drug-target interaction are not well understood. We studied the molecular mechanisms of gemcitabine-induced apoptosis and determined the role of p53 function on the cytotoxic effects of gemcitabine in human nonsmall cell lung cancer (NSCLC) H1299 and H1299/p53 cells. Here, we found that gemcitabine induced an apoptotic cell death via a Bcl-2-dependent caspase-9 activation pathway. Moreover, phosphorylated activation of extracellular signal-regulated kinase (ERK) was observed upon gemcitabine treatment. Genetical or pharmacological inhibition of ERK activation markedly blocked gemcitabine-induced cell death. Furthermore, inactivation of Akt was also involved in this event. Taken together, our observations indicate that ERK activation and Akt inactivation mediated gemcitabine-induced apoptosis independently of p53 in human NSCLC H1299 cells.

By combining suppression subtractive hybridization and microarray to examine gene expressions between metastatic and non-metastatic non-small cell lung cancer (NSCLC), we have identified differential expression spectra of matrix metalloproteinases (MMP). Among MMPs, expressions of MMP-13, -14, -15 and -24 decreased, those of MMP-9, -11, -12, -16, -17, -19 and -23B did not change, and those of MMP-1, -2, -7, -8 and -10 increased dramatically. Overexpressions of MMP-1, -2, -7 and -10 were confirmed by reverse transcription-polymerase chain reaction. In this study we further assessed the clinical significance of MMP-1, -2, -7 and -10. Specimens from 472 patients with completely resected NSCLC were examined by immunohistochemistry. The median follow-up period was 38 months (range, 2-113 months). Overexpression of MMP-1 was observed in 72.9% (n=344) of 472 patients, that of MMP-2 was 77.9% (n=352), MMP-7 63.3% (n=299) and that of MMP-10 was 27.1% (n=128). For patients with lymph node metastasis, MMP-1 and -2 overexpressions were not only independent prognostic factors for unfavorable outcome, but also associated with decreased survival (p=0.0015, and p=0.011 respectively). The present study showed that MMP expression spectrum in NSCLC was heterogeneous: expression of some MMP increased, some unchanged, while some decreased. Therefore, it should be worth determining MMP expression pattern as a regimen reference for NSCLC patients who were scheduled to receive MMP inhibitor, which was class-specific, as adjuvant therapeutic agent.

Epithelial growth factor receptor (EGFR) has been proposed as a target for anticancer therapy. ZD1839 (Iressa) is a quinazoline derivative that selectively inhibits the EGFR tyrosine kinase activity and is under clinical use in cancer patients. However, the molecular mechanisms involved in ZD1839-mediated anticancer effects remain largely uncharacterized. In this study, exposure of human lung adenocarcinoma A549 cells to ZD1839 caused G1 arrest, and subsequently induced apoptosis. Moreover, ZD1839 increased the protein levels of p27(KIP1) and retinoblastoma-related Rb2/p130 while decreased the expression of cyclin-dependent kinase-2 (CDK2), CDK4, CDK6 and cyclin-D1, cyclin-D3. In vitro kinase assay showed that ZD1839 decreased these CDKs expression in A549 cells, leading to significantly reduce their kinase activities. In addition, ZD1839-induced death of A549 cells with characteristics of apoptosis including apoptotic morphological changes, DNA fragmentation and enhancement of TUNEL-positive cell. These events were accompanied by a marked increase of Fas protein expression, and activation of caspase-2, -3, -8. Co-treatment of cells with Fas antagonist antibody significantly blocked ZD1839-induced apoptosis. Caspase-8 and caspase-3 inhibitors, but not a caspase-9 inhibitor, were also capable of restoring cell viability. Our results indicate that downregulation of the expression and function of CDK2, CDK4, CDK6, cyclin-D1 and cyclin-D3, as well as upregulation of p27(KIP1) and pRb2/p130, are strong candidates for the cell cycle regulator that arrests ZD1839-treated A549 cells at G1 phase. Furthermore, upregulation of Fas appears to play a major role in the initiation of ZD1839-induced apoptosis, activation of caspase-8/caspase-3 cascade is involved in the execution phase of this death program.

The relative contribution of promoter hypermethylation and aberrant splicing to the inactivation of the fragile histidine triad (FHIT) gene is unclear. Using genetic and epigenetic analyses, the current investigation examines the loss of protein and mRNA expression, and 5'CpG hypermethylation and allelic imbalance of the FHIT gene in a series of 129 non-small cell lung cancer (NSCLC) samples, in parallel with clinicopathological analyses. We found that 50% of NSCLC patients had aberrant protein expression, which was more frequent in squamous cell carcinomas (SQ) (69%) than in adenocarcinomas (AD) (28%) (P < 0.0001). 5'CpG hypermethylation of FHIT was identified in 31% of patients. Abnormally-sized FHIT transcripts were also observed in 24% of patients and were attributed to various exonic deletions, mainly in the region of exons 4-8. Allelic imbalance of the FHIT locus and its correlation with the status of Fhit expression, 5'CpG hypermethylation, and aberrant splicing, indicated that biallelic inactivation of Fhit expression could be induced by 5'CpG hypermethylation of one allele and alternative splicing in the other allele. Moreover, an 83% concordance in the methylation status of FHIT was demonstrated between 12 samples of bronchial precancerous lesions taken before surgery and their matched resected tumours. Our data suggest that FHIT 5'CpG hypermethylation and splicing alterations are both predominant mechanisms involved in the aberrant expression of the FHIT gene, and that FHIT 5'CpG methylation may be potentially used as a supplemental detection marker for NSCLC.

Objective: To establish a new model for analyzing the correlation between the terminal restriction fragment (TRF) length and telomerase activity in non-small cell lung cancer (NSCLC) patients due to inconsistent results in previous reports., Methods: Between January 1999 and December 1999, 79 NSCLC patients were studied. The activity of telomerase was measured by telomeric repeat amplification protocol, and the telomere restriction fragment (TRF) length was measured by Telomere Length Assay Kit and Southern blotting. The correlation between expression of telomerase activity and the TRF length ratio (TRFLR) using the tumor-to-normal TRFLR (t/n TRFLR) was examined., Results: Positive telomerase activity was observed in 48 (60.8%) of the tumor tissue samples and in 5 (6.3%) of the normal tissue samples (P < 0.0001). The mean TRF lengths were 5.32+/-1.53 kb in normal tissue samples and 3.95+/-1.92 kb in tumor tissue samples, respectively (P = 0.0001). The 4-year cumulative survival rates of lower t/n TRFLR (< or = 75%) and higher t/n TRFLR (>75%) patients were 69.2 and 41.2%, respectively (P = 0.0227). Independent prognostic factors include t/n TRFLR (P = 0.014), T-status (P = 0.027), and N-status (P = 0.020) of the tumor., Conclusions: Our data suggest that there is a good correlation between the t/n TRFLR and expression of telomerase activity. A higher t/n TRFLR may indicate that the tumor regains its ability to repair the telomere lost and escapes the apoptosis scenario, which is subsequently reflected in poorer patient outcomes.

Purpose and Experimental Design: The molecular mechanisms by which the p14ARF gene is altered in non-small cell lung cancer (NSCLC) are complex and unclear. Using genetic and epigenetic analyses, we examined various molecular alterations including the loss of protein and mRNA expression, and 5'CpG hypermethylation, allelic imbalance, and mutation of the p14ARF gene in a series of 102 NSCLC samples, in parallel with clinicopathological and prognostic analyses. To clarify the biological significance of p14ARF alterations, its relationship with p16INK4a and p53 alterations was also examined., Results: We found that 34% of NSCLC patients had aberrant P14ARF protein expression, which was more frequent in adenocarcinomas (AD; 44%) than in squamous cell carcinomas (22%; P = 0.024). A high concordance was observed between alterations in protein and mRNA expression and 5'CpG hypermethylation (P

Background: The divergences in the clinical significance of bone marrow microinvolvement (BMM) in patients with nonsmall cell lung carcinoma (NSCLC) necessitated a long-term large series study., Methods: Between March 1997 and June 2001, the authors analyzed 212 bone marrow specimens (from the posterior iliac crest) taken from patients with NSCLC before surgery. The degree of tumor differentiation included well differentiated carcinoma in 12 Patients, moderately differentiated carcinoma in 112 Patients, and poorly differentiated carcinoma in 68 Patients. The pTNM staging (according the the criteria of the American Joint Committee on Cancer) included Stage IA in 8 patients, Stage IB in 70 patients, Stage IIB in 36 patients, Stage IIIA in 54 patients, Stage IIIB in 14 patients, and Stage IV in 10 patients. The specimens were evaluated by immunohistochemical staining with antihuman cytokeratin AE1/AE3, Ber-EP4, and clone MNF116 mixed solution to detect the presence of malignant epithelial cells in the bone marrow., Results: Positive results were observed in 66 patients (34.4%). The occurrence of BMM was not found to be related to patient age, gender, cell type, or TNM status. The 5-year disease-free survival rates were 44.9% and 40.5% in BMM-negative and BMM-positive patients, respectively (P = 0.3797). The 5-year cumulative survival rates were 43.5% and 44.0% in BMM-negative and BMM-positive patients, respectively (P = 0.4262). Multivariate analysis failed to demonstrate BMM as an independent prognostic factor (P = 0.1817)., Conclusions: The results of the current study showed that although BMM was observed frequently in patients with NSCLC, regardless of tumor stage and pathologic types, its occurrence was not a good predictor of long-term prognosis., (Copyright 2004 American Cancer Society.)

Objective: The management of ipsilateral multifocal non-small cell lung cancer (NSCLC) in different lobes is still controversial. We analyzed our surgical results and the prognostic factors with the findings of other studies and evaluated the surgical feasibility., Methods: Between January 1, 1983 and December 31, 2001, 1408 patients underwent operation for primary NSCLC, including 20 patients who received complete resections for multifocal NSCLC of the same histological type in ipsilateral different lobes., Results: The 1-, 2- and 5-year survival rate of the 20 patients were 60.0, 39.3 and 28.1%, respectively. There were no statistically significant differences in T-status, gender, pathological type, and stage. An excellent 5-year survival rate of 66.7% (median, 101 months) in the group without node involvement was found (N0 vs. N1+2, P=0.0872)., Conclusion: Our data suggest that surgical resection is mandatory in patients with ipsilateral multifocal NSCLC when there is no evidence of node metastasis.

Objective: We evaluate the clinical results of thoracoscopic T3-4 sympathicotomy for axillary osmidrosis., Methods: From July 1995 to June 2001, 262 patients (208 females, 54 males) with axillary osmidrosis have been treated by thoracoscopic T3-4 sympathicotomy. All patients were followed up for a minimum of 10 months (average 42 months). The patients were evaluated by telephone or mail questionnaires. The results were categorized as excellent, good, fair, or poor., Results: There were no surgical mortalities and major complications in this series. The surgical outcomes were excellent in 144 (55%) patients, good in 39 (15%) patients, fair in 55 (21%) patients, and poor in 24 (9%) patients. Compensatory sweating developed in 171 (65%) patients. Dry hands developed in 40 (15%) patients., Conclusions: Thoracoscopic T3-4 sympathicotomy is a safe, fast, cosmetic, and effective method in treating axillary osmidrosis.

Many cancer and immortal cells exhibit telomerase activity that stabilizes telomere lengths, possibly contributing to cell immortality and carcinogenesis. The aim of this study was to elucidate the clinicopathological relationship between telomerase activity and telomerase reverse transcriptase subunit (hTERT) status in non small cell lung cancer. hTERT status in non small cell lung cancer using telomeric repeat amplification protocol (TRAP) and RT-PCR assay, respectively. Telomerase activity and hTERT were detected in 85.7 and 80.3% of cancerous tissues, respectively. Telomerase activity does not correlate with clinicopathological variables. However, there was an association between p53-correlated expression and hTERT negative status. Lung cancer patients without telomerase activity survived for a significantly longer period than those with telomerase activity. In addition, hTERT was not associated with the prognosis. TERT expression did not correlate well with any clinical parameter. Reactivated telomerase activity may be a poor prognostic factor in NSCLCs.

We assessed the association of three genetic polymorphisms, NAD(P)H quinone oxidoreductase (NQO1), Glutathione-S-transferase P1 (GSTP1), and manganese superoxide dismutase (MnSOD), with lung cancer risk in 198 cases and 332 controls in Taiwan. Overall, NQO1 and MnSOD polymorphisms were not associated with an increased risk of lung cancer. Individuals carrying variant alleles of GSTP1 were at higher risk of squamous cell lung carcinoma (odds ratio, 1.63; 95% confidence interval, 0.96-2.74). When the groups were further stratified by smoking status following gender and histological type, the wild-type NQO1 was associated with lung adenocarcinoma among smokers but not among nonsmokers (odds ratio, 2.49; 95% confidence interval, 1.17-5.32). These results suggest that NQO1 plays a role in the development of cigarette smoking-associated lung adenocarcinoma. In addition, GSTP1 polymorphism was associated with the risk of squamous cell lung carcinoma in Taiwan.

We performed a genetic and epigenetic study of the hMLH1 and hMSH2 mismatch repair genes in resected primary tumors from 77 non-small cell lung cancer (NSCLC) patients. The molecular alterations examined included the loss of mRNA and protein expression as well as promoter methylation, and the allelic imbalance of the chromosomal regions that harbor the genes. We found that 78% and 26% of patients showed at least one type of molecular alteration within the hMLH1 and hMSH2 genes, respectively. Promoter methylation of the hMLH1 gene was present in 55.8% of tumors, and was significantly associated with the reduction in mRNA and protein expression (P = 0.001). A 72% concordance of aberrant methylation in sputum samples with matched resected tumors was found. In addition, a 93% consistency between the promoter methylation and the mRNA expression of the hMSH2 gene was found in 14 female NSCLC patients. However, no correlation was found between the expression of hMLH1 and hMSH2 proteins and the allelic imbalance of five microsatellite markers closely linked to the genes. Our results suggest that hMLH1 is the major altered mismatch repair gene involved in NSCLC tumorigenesis, and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMLH1 gene may be identified in sputum samples to serve as a potential diagnostic marker of NSCLC.

Aims: The present study retrospectively examines the expression of caspase-3 in primary resected esophageal squamous cell carcinoma (ESCC) and the correlation between the outcome of patients and the expression of proteins., Methods: Immunohistochemistry and Western blot analyses were used to analyse the expression of caspase-3 in 40 archival specimens of patients with primary resected ESCC., Results: According to our cut-off point of the staining for caspase-3, 24 (60%) cases were positive and 16 (40%) negative. Caspase-3 expression correlated with a significant favorable prognosis in primary resected ESCC (P=0.02). A multivariate analysis of clinical and biological factors indicated that stage, tumor differentiation, and caspase-3 expression were independent prognostic factors., Conclusions: Caspase-3 expression might be a good and new prognostic indicator for primary resected ESCC.

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender-related changes in non-small cell lung cancer (NSCLC). We investigated smoking-related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RARbeta, CDH13, MGMT and GSTP1]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RARbeta were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender-related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography-related differences in the methylation profiles of NSCLC tumors., (Copyright 2002 Wiley-Liss, Inc.)

Recently, by using differential display on specimens of non-small cell lung cancer (NSCLC), we detected overexpression of dihydrodiol dehydrogenase (DDH) that was rarely expressed in the corresponding normal lung tissue. DDH overexpression was correlated with poor prognosis of patients with advanced NSCLC. Because DDH could metabolize polycyclic aromatic hydrocarbons (PAH) in the liver, DDH overexpression in NSCLC would suggest an association with carcinogenesis and disease progression. In this study, we investigated DDH expression in 103 patients with resected stage I NSCLC. Expression of DDH was detected by using immunohistochemistry. Relation between DDH expression and clinicopathological parameter (age, gender, smoking habit, tumor status, histological type, cell differentiation, local recurrence, distant metastasis or survival) was analyzed by statistical analysis. DDH overexpression was detected in 39 (37.9%) of 103 pathological sections. Frequency of DDH overexpression was significantly higher in male patients (p=0.043) and patients with squamous cell carcinoma (p<0.005). Among 103 patients, 14 patients had local recurrence and 28 patients had distant metastasis during follow-up examination. The 5-year survival rate of these patients was poorer than those who did not have local recurrence or distant metastasis (both were p<0.005, respectively). Although patients with low DDH expression had more favorable outcome than those with DDH overexpression, in terms of survival rate no statistical significance was detected (p=0.889). The results suggest that DDH expression may serve as an early but not prognostic biomarker for patients with resectable stage I NSCLC.

Recently, we reported that p16 protein expression was nondetectable in 49.5% of 107 resected nonsmall cell lung cancers (NSCLCs), suggesting that the p16(INK4a) gene is frequently inactivated in primary NSCLC. To identify the molecular basis for this p16 immunohistochemical negativity further, we performed a genetic and epigenetic study of p16(INK4a) status in a series of 115 NSCLC samples parallel to the clinicopathologic and prognostic analyses. Microdissected tumor DNA samples were screened for homozygous deletion using comparative multiplex-polymerase chain reaction (PCR), for intragenic mutation using direct sequencing and for loss of heterozygosity (LOH) using an intragenic microsatellite marker, D9S942. Of these samples, 67 were further analyzed by SmaI-based PCR methylation assay to evaluate aberrant methylation at the gene. To examine the correlation of aberrant methylation in tumor and sputum samples, sputum samples from 12 matched patients were assessed for this change. We found that methylation of the p16(INK4a) gene was present in 38 of the 67 (56.7%) tumors and was significantly associated with negative p16 protein expression (p = 0.029). A 92% (11/12) concordance of sputum samples with matched resected tumors was found. The survival rates among adenocarcinoma patients with p16(INK4a) methylation were lower, but at a level of borderline significance compared with those patients without methylation (p = 0.071). In addition, 29.4% of the informative cases were found to harbor LOH at D9S942. None of the 115 microdissected tumors exhibited homozygous deletion in the p16(INK4a) gene. Only 1 patient exhibited a complex mutation at the fourth ankyrin repeat consensus sequence and concordantly demonstrated p16 immunohistochemical negativity. Overall, 69% (79/115) of NSCLC tumors had at least 1 type of p16(INK4a) alteration. Our data provide compelling evidence that p16(INK4a) alterations are involved in NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in p16(INK4a) deregulation., (Copyright 2002 Wiley-Liss, Inc.)

To investigate the etiological association of allelic loss at chromosomal regions containing tumor suppressor genes (TSGs) in non-small cell lung cancer (NSCLC) in Taiwan, we examined 48 microdissected NSCLC samples for loss of heterozygosity (LOH) at nine loci where TSGs are localized nearby. The associations of LOH at each locus with clinicoparameters and prognosis were also examined. The frequent LOH was observed using markers, D3S1285 near the FHIT gene (58.3%), D17S938 near the p53 gene (56.7%), D9S925 near the p16 gene (54.5%), and D13S153 near the RB gene (47.6%). The occurrence of LOH at each TSG locus was compared with the patients' clinicoparameters. The incidence of LOH at D17S938 (p53 gene) and D3S4545 (VHL gene) was significantly higher in squamous carcinoma tumors than in adenocarcinoma tumors (P = 0.003 and 0.024, respectively). LOH of these two loci also occurred frequently in tumors from smoker patients compared to that from nonsmoker patients (P = 0.013 and 0.025, respectively). LOH at D13S153 (RB gene) was also associated with smoking (P = 0.008). In addition, the prognostic analyses indicated that the patients with LOH at D18S535 (18q21, near the SMAD2/4 gene) had significantly longer post-operative survival time compared to those without LOH (P = 0.03). Our results suggested that LOH at FHIT, p53, and p16 genes may occur frequently in NSCLC patients in Taiwan. In addition, LOH at p53, RB, and VHL may associate with smoking or squamous carcinoma patients and LOH at SMAD2/4 may be correlated with better prognosis.