[Show abstract][Hide abstract] ABSTRACT: Pemetrexed is approved for first-line and maintenance treatment of patients with advanced or metastatic non-small-cell lung cancer (NSCLC). The protein kinase Akt/protein kinase B is a well-known regulator of cell survival which is activated by pemetrexed, but its role in pemetrexed-mediated cell death and its molecular mechanisms are unclear. This study showed that stimulation with pemetrexed induced S-phase arrest and cell apoptosis and a parallel increase in sustained Akt phosphorylation and nuclear accumulation in the NSCLC A549 cell line. Inhibition of Akt expression by Akt specific siRNA blocked S-phase arrest and protected cells from apoptosis, indicating an unexpected proapoptotic role of Akt in the pemetrexed-mediated toxicity. Treatment of A549 cells with pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and Ly294002, similarly inhibited pemetrexed-induced S-phase arrest and apoptosis and Akt phosphorylation, indicating that PI3K is an upstream mediator of Akt and is involved in pemetrexed-mediated cell death. Previously, we identified cyclin A-associated cyclin-dependent kinase 2 (Cdk2) as the principal kinase that was required for pemetrexed-induced S-phase arrest and apoptosis. The current study showed that inhibition of Akt function and expression by pharmacological inhibitors as well as Akt siRNA drastically inhibited cyclin A/Cdk2 activation. These pemetrexed-mediated biological and molecular events were also observed in a H1299 cell line. Overall, our results indicate that, in contrast to its normal prosurvival role, the activated Akt plays a proapoptotic role in pemetrexed-mediated S-phase arrest and cell death through a mechanism that involves Cdk2/cyclin A activation.
PLoS ONE 01/2014; 9(5):e97888. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor cells before and after epidermal growth-factor receptor (EGFR) tyrosine-kinase inhibitor (TKI) therapy might display different characteristics. The aim of this study was to evaluate the influence of prior EGFR TKI therapy on the efficacy of subsequent pemetrexed plus platinum (PP) in advanced chemonaïve patients with EGFR-mutant lung adenocarcinoma.
OncoTargets and Therapy 01/2014; 7:799-805. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Luteolin is a natural flavonoid that possesses a variety of pharmacological activities, such as anti-inflammatory and anti-cancer abilities. Whether luteolin regulates the transformation ability of Lung cancer cells remains unclear. The current study aims to uncover the effects and underling mechanisms of luteolin in regulation of and Epithelial-mesenchymal transition of lung cancer cells.
The lung adenocarcinoma A549 cells were used in this experiment; the cells were pretreated with luteolin followed by administration with TGF-β1. The expression levels of various cadherin and related upstream regulatory modules were examined, KEY FINDINGS: Pretreatment of luteolin prevented the morphological change and downregulation of E-cadherin of A549 cells induced by TGF-β1. In addition, the activation of PI3K-AKT-IκBa-NF-κB-snail pathway which leading to the decline of E-cadherin induced by TGF-β1 also attenuated under the pretreatment of luteolin.
We provide the mechanisms about how luteolin attenuated the Epithelial-mesenchymal transition of A549 lung cancer cells induced by TGF-β1. This finding will strengthen the anti-cancer effects of flavonoid compounds via the regulation of migration/invasion and EMT ability of various cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts.
Evidence-based Complementary and Alternative Medicine 01/2013; 2013:613950. · 1.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pemetrexed (MTA) is a multitargeted antifolate drug approved for lung cancer therapy. Clinically, supplementation with high doses of folic acid (FA) and vitamin B12 (VB12) lowers MTA cytotoxicities. An antagonistic effect of FA/VB12 on MTA efficacy has been proposed. However, patients who receive FA/VB12 show better tolerance to MTA with improved survival. The aims of this study are to investigate the modulation of FA and VB12 on MTA drug efficacy in human nonsmall cell lung cancer (NSCLC) cell lines. The sensitivities of cells, apoptosis, and MTA-regulated proteins were characterized to determine the possible effects of high doses of FA and VB12 on MTA efficacy. MTA has the lowest efficacy under 10% serum conditions. However, supplementation with FA and VB12 individually and additively reversed the insensitivity of NSCLC cells to MTA treatment with 10% serum. The enhanced sensitivities of cells following FA/VB12 treatment were correlated with increasing apoptosis and were specific to MTA but not to 5-fluorouracil (5-FU). Enhanced sensitivity was also associated with p21(WAF1/Cip1) expression level. Our results revealed no antagonistic effect of high doses of FA/VB12 on MTA efficacy in cancer cells grown in nutrient medium. Furthermore, these data may partially explain why supplementation of FA and VB12 resulted in better survival in MTA-treated patients.
BioMed research international. 01/2013; 2013:389046.
[Show abstract][Hide abstract] ABSTRACT: To identify common genetic variants that contribute to lung cancer susceptibility, we conducted a multistage genome-wide association study of lung cancer in Asian women who never smoked. We scanned 5,510 never-smoking female lung cancer cases and 4,544 controls drawn from 14 studies from mainland China, South Korea, Japan, Singapore, Taiwan and Hong Kong. We genotyped the most promising variants (associated at P < 5 × 10(-6)) in an additional 1,099 cases and 2,913 controls. We identified three new susceptibility loci at 10q25.2 (rs7086803, P = 3.54 × 10(-18)), 6q22.2 (rs9387478, P = 4.14 × 10(-10)) and 6p21.32 (rs2395185, P = 9.51 × 10(-9)). We also confirmed associations reported for loci at 5p15.33 and 3q28 and a recently reported finding at 17q24.3. We observed no evidence of association for lung cancer at 15q25 in never-smoking women in Asia, providing strong evidence that this locus is not associated with lung cancer independent of smoking.
[Show abstract][Hide abstract] ABSTRACT: p53 plays an important role in mitotic checkpoint, but what its role is remains enigmatic. Aurora A is a Ser/Thr kinase involved in correcting progression of mitosis. Here, we show that p53 is a negative regulator for Aurora A. We found that p53 deficiency leads to Aurora A elevation. Ectopic expression of p53 or DNA damage-induced expression of p53 can suppress the expression of Aurora A. Mechanistic studies show that p53 is a negative regulator for Aurora A expression through both transcriptional and posttranslational regulation. p53 knockdown in cancer cells reduces the level of p21, which, in turn, increases the activity of CDK2 followed by induction of Rb1 hyperphosphorylation and its dissociation with transcriptional factor E2F3. E2F3 can bind to Aurora A gene promoter, potentiating Aurora A gene expression and p53 deficiency, enhancing the binding of E2F3 on Aurora A promoter. Also, p53 deficiency leads to decelerating Aurora A's turnover rate, due to the fact that p53 deficiency causes the downregulation of Fbw7α, a component of E3 ligase of Aurora A. Consistently, p53 knockdown-mediated Aurora A elevation is mitigated when Fbw7α is ectopically expressed. Thus, p53-mediated Aurora A degradation requires Fbw7α expression. Significantly, inverse correlation between p53 and Aurora A elevation is translated into the deregulation of centrosome amplification. p53 knockdown leads to high percentages of cells with abnormal amplification of centrosome. These data suggest that p53 is an important negative regulator of Aurora A, and that loss of p53 in many types of cancer could lead to abnormal elevation of Aurora A and dysregulated mitosis, which provides a growth advantage for cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Aurora B is a mitotic checkpoint kinase that plays a pivotal role in the cell cycle, ensuring correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in many types of human cancers, which has made it an attractive target for cancer therapies. Tumor suppressor p53 is a genome guardian and important negative regulator of the cell cycle. Whether Aurora B and p53 are coordinately regulated during the cell cycle is not known. We report that Aurora B directly interacts with p53 at different subcellular localizations and during different phases of the cell cycle (for instance, at the nucleus in interphase and the centromeres in prometaphase of mitosis). We show that Aurora B phosphorylates p53 at S183, T211, and S215 to accelerate the degradation of p53 through the polyubiquitination-proteasome pathway, thus functionally suppressing the expression of p53 target genes involved in cell cycle inhibition and apoptosis (e.g., p21 and PUMA). Pharmacologic inhibition of Aurora B in cancer cells with WT p53 increased p53 protein level and expression of p53 target genes to inhibit tumor growth. Together, these results define a mechanism of p53 inactivation during the cell cycle and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise the tumor suppressor function of p53. We have elucidated the antineoplastic mechanism for Aurora B kinase inhibitors in cancer cells with WT p53.
Proceedings of the National Academy of Sciences 05/2012; 109(24):E1513-22. · 9.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We explored potential associations between genetic polymorphisms in genes related to DNA repair and detoxification metabolism and epidermal growth factor receptor (EGFR) mutations in a cohort of 410 never-smoking patients with lung adenocarcinoma. Multivariate-adjusted odds ratios (aORs) and corresponding 95% confidence intervals (CI) of EGFR mutation status in association with the genotypes of DNA repair and detoxification metabolism genes were evaluated using logistic regression analysis. We found an association between in-frame deletion in EGFR exon 19 and a single nucleotide polymorphism (SNP) rs1800566C/T located in NQO1 (aOR, 2.2 with 95% CI, 1.0-4.8) in female never-smokers. The SNP rs744154C/G in ERCC4 was also associated with the EGFR exon 19 in-frame deletion both in never-smokers (aOR, 1.7 with 95% CI, 1.0-3.0) and female never-smokers (aOR, 1.9 with 95% CI, 1.0-3.6). Although the association was marginally significant in multivariate logistic regression analysis, the A/A genotype of rs1047840 in EXO1 was associated with a 7.6-fold increase in the occurrence of the EGFR exon 19 in-frame deletion in female never-smokers. Moreover, risk alleles in NQO1, ERCC4 and EXO1 were associated with an increasing aOR of the EGFR exon 19 in-frame deletion both in never-smokers (p = 0.007 for trend) and female never-smokers (p = 0.002 for trend). Our findings suggest that the in-frame deletion in EGFR exon 19 is associated with polymorphisms in DNA repair and detoxification metabolism genes in never-smoking lung adenocarcinoma patients, especially in females.
International Journal of Cancer 05/2012; · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effective targeted therapy for lung squamous cell carcinoma (SCC) is needed. The epidermal growth factor receptor (EGFR) mutation rate is low in lung SCC. The aim of this study was to evaluate the status of erlotinib treatment and EGFR mutation in lung SCC patients.
We retrospectively enrolled lung cancer patients with SCC histology and history of erlotinib treatment. The primary objective was to assess overall response rate (ORR) and disease control rate (DCR) and the secondary objective was to assess progression-free survival (PFS) and overall survival (OS). EGFR mutations were assessed in parts of patients using both direct sequencing and protein nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp methods.
In total, 92 patients were analyzed (75 men and 17 women, median age 69 years, and 74 current or former smokers). Sixteen patients achieved partial response and 9 had stable disease. The ORR was 17.4% and the DCR was 27.2%. The PFS and OS were longer in patients with disease control than with progressive disease (PFS 7.8 versus 1.3 months and OS 20.7 versus 2.7 months, both p<0.0001). The 1-year survival rate was 21.7%. In 27 patients with adequate specimens for molecular analysis (including 4 PR and 4 SD), two (7.4%) had EGFR complex mutations. One patient experienced response to erlotinib and the other did not.
A significant proportion of lung SCC patients would derive a clinical benefit from erlotinib treatment. The relatively higher response rate than the EGFR mutation rate in present study needs further evaluation.
Lung cancer (Amsterdam, Netherlands) 03/2012; 77(1):128-33. · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A recent genome-wide association study (GWAS) of subjects from Japan and South Korea reported a novel association between the TP63 locus on chromosome 3q28 and risk of lung adenocarcinoma (p = 7.3 × 10(-12)); however, this association did not achieve genome-wide significance (p ≤ 10(-7)) among never-smoking males or females. To determine if this association with lung cancer risk is independent of tobacco use, we genotyped the TP63 SNPs reported by the previous GWAS (rs10937405 and rs4488809) in 3,467 never-smoking female lung cancer cases and 3,787 never-smoking female controls from 10 studies conducted in Taiwan, Mainland China, South Korea, and Singapore. Genetic variation in rs10937405 was associated with risk of lung adenocarcinoma [n = 2,529 cases; p = 7.1 × 10(-8); allelic risk = 0.80, 95% confidence interval (CI) = 0.74-0.87]. There was also evidence of association with squamous cell carcinoma of the lung (n = 302 cases; p = 0.037; allelic risk = 0.82, 95% CI = 0.67-0.99). Our findings provide strong evidence that genetic variation in TP63 is associated with the risk of lung adenocarcinoma among Asian females in the absence of tobacco smoking.
Human Genetics 02/2012; 131(7):1197-203. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pemetrexed, a multitargeted antifolate with the ability to inhibit several enzymes involved in purine and pyrimidine syntheses, has demonstrated clinical activity in non-small cell lung cancer cells, as well as in a broad array of other solid tumors. In this study, we show that inducing cell cycle S-phase arrest and apoptosis in human lung adenocarcinoma A549 cells with pemetrexed is associated with increased cyclin-A and cyclin-dependent kinase 2 (Cdk2) protein and Cdk2/cyclin-A kinase activity. Knockdown of cyclin-A using small interfering RNA (siRNA), and inhibiting Cdk2 activity with flavopiridol, strikingly reduced S-phase arrest and apoptosis. Moreover, pemetrexed induced sustained activation of extracellular signal-regulated kinase1/2 (ERK1/2). Knockdown of ERK1/2 using specific siRNA, as well as known inhibitors (PD98059 and U0126), effectively suppressed the expression of cyclin-A and Cdk2, and reduced S-phase arrest and apoptosis induced by pemetrexed. These data provide the first evidence that pemetrexed-induced S-phase arrest and apoptosis is associated with an increase in Cdk2 and cyclin-A expression and activation, which is ERK-dependent and upstream of caspase-3. Our findings suggest that the ERK-mediated Cdk2/cyclin-A signaling pathway is an important regulator of pemetrexed-induced S-phase arrest and apoptotic cell death.
European journal of pharmacology 08/2011; 663(1-3):17-26. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been proven more effective for patients with lung adenocarcinoma with EGFR-activating mutation rather than wild type, the former group still includes approximately 30% nonresponders. The molecular basis of this substantial response heterogeneity is unknown. Our purpose was to seek molecular aberrations contributing to disease progression at the genome-wide level and identify the prognostic signature unique to patients with EGFR-activating mutation.
We first investigated the molecular differences between tumors with EGFR-activating mutation and wild-type tumors by conducting high-density array comparative genomic hybridization on a collection of 138 adenocarcinoma tissues. We then used an independent group of 114 patients to validate the clinical relevance of copy-number alterations (CNAs) in predicting overall and disease-free survival. Finally, focusing on 23 patients with EGFR mutation receiving EGFR-TKI treatment, we investigated the association between CNAs and response to EGFR-TKIs.
We identified chromosome regions with differential CNAs between tumors with EGFR-activating mutation and wild-type tumors and found the aberration sites to cluster highly on chromosome 7p. A cluster of six representative chromosome 7p genes predicted overall and disease-free survival for patients with EGFR-activating mutation but not for those with wild type. Importantly, simultaneous presence of more genes with increased CNAs in this cluster correlated with less favorable response to EGFR-TKIs in patients with EGFR-activating mutation.
Our results shed light on why responses to EGFR-TKIs are heterogeneous among patients with EGFR-activating mutation. They may lead to better patient management in this population.
Journal of Clinical Oncology 08/2011; 29(25):3435-42. · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In terms of drug resistance, cancer cells usually benefit from high clusterin (CLU) expression on chemotherapy. In contrast, CLU expression has been found to be a favorable prognostic factor in lung cancer patients. The aims of this study are to determine the association between CLU expression and chemotherapeutic sensitivity and the potential role of CLU in migration in human non-small-cell lung cancer (NSCLC) cell lines.
The levels of clusterin in NSCLC cell lines were altered by short hairpin RNA interference (shRNAi) and overexpression on chemosensitivity assay. Migratory ability of these cell lines was also investigated.
H1355 cells with the highest level of CLU demonstrated the lowest sensitivities to Adriamycin (ADR), docetaxel (DOC), and gemcitabine (GEM) treatment. Inhibition of CLU expression in H1355 cells resulted in higher chemosensitivities. When CLU was stably overexpressed in A549 and H1299 cells, only the chemosensitivity to ADR was reduced. The migratory ability of CLU-overexpressing cells significantly decreased. Moreover, MMP2 transcription was inhibited in CLU-overexpressing H1299 cells. These results indicated lower metastatic potential for cancer cells with high CLU level.
Lung cancer cells with high level of CLU have reduced chemosensitivity. High level of CLU may result in migratory inhibition and thus favorable prognosis in lung cancer.
Cancer Chemotherapy and Pharmacology 06/2011; 69(1):145-54. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Early lung adenocarcinoma may present with ground-glass opacity (GGO) component in computed tomography (CT) scan. Epidermal growth factor receptor (EGFR) mutation had been reported in patients with lung cancer with GGO patterns. Nevertheless, the correlation between clinical characteristics, CT image patterns, and EGFR mutation status was indeterminate.
Patients with stage I lung adenocarcinoma with tumor lesions less than 3 cm were included and classified into pure GGO, part-solid, and solid patterns by CT scan images. All patients had EGFR mutation test from frozen tumors. Available paraffin-embedded archival tissues were microdissected into three different locations similar to CT images with central and peripheral parts of tumor, and adjacent normal part for EGFR mutation tests.
Totally, 162 patients were analyzed, 90 women and 72 men, and 128 nonsmokers. The patients included 35 (21.6%) pure GGO, 41 (25.3%) part-solid, and 86 (53.1%) solid lesions. The EGFR mutation rate was 64.2% (n = 104). Analysis of the correlation between CT image patterns and EGFR mutation, the less GGO ratio had more typical mutation, especially L858R (p = 0.037). In 45 microdissected tumors, the central and peripheral parts had the same EGFR mutation status. In adjacent normal parts, 5 of 32 (15.6%) EGFR mutant patients had identical mutation but none in nonmutant patients.
In stage I lung adenocarcinoma, typical mutation, especially L858R was detected more frequent in invasive solid pattern and significantly less in pure GGO pattern. EGFR mutation is an early event in the pathogenesis of lung adenocarcinoma and may facilitate the tumor into aggressive behavior.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 06/2011; 6(6):1066-72. · 4.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess whether polymorphisms of genes related to estrogen biosynthesis and metabolism are associated with EGFR mutations.
We studied 617 patients with lung adenocarcinoma, including 302 never-smoking women. On the basis of multiple candidate genes approach, the effects of polymorphisms of CYP17, CYP19A1, ERα, and COMT in association with the occurrence of EGFR mutations were evaluated using logistic regression analysis.
In female never-smokers, significant associations with EGFR L858R mutation were found for the tetranucleotide (TTTA)(n) repeats in CYP19A1 (odds ratio, 2.6; 95%CI, 1.2-5.7 for 1 or 2 alleles with (TTTA)(n) repeats >7 compared with both alleles with (TTTA)(n) repeats ≤ 7), and the rs2234693 in ERα (OR, 2.1; 95% CI, 1.1-4.0 for C/T and C/C genotypes compared with T/T genotype). The C/C genotype (vs. T/T genotype) of ERα was significantly associated with EGFR L858R mutation (OR, 3.0; 95% CI, 1.1-8.1), in-frame deletion (OR, 2.9; 95% CI, 1.1-7.6) and other mutations (OR, 4.3; 95% CI, 1.3-14.0). The genotype of COMT rs4680 was significantly associated with EGFR L858R mutation in female and male never-smokers showing OR's (95% CI) of 1.8 (1.0-3.2) and 3.6 (1.1-11.3), respectively, for genotypes G/A and G/G compared with genotype A/A. The number of risk alleles of CYP17, CYP19A1, ERα, and COMT was associated with an increasing OR of EGFR L858R mutation in female never-smokers (P = 0.0002 for trend).
The L858R mutation of EGFR is associated with polymorphisms of genes related to estrogen biosynthesis and metabolism in never-smoking female lung adenocarcinoma patients.
Clinical Cancer Research 02/2011; 17(8):2149-58. · 7.84 Impact Factor