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OBJECTIVES: E-cigarettes are the most commonly used nicotine containing products among youth. In vitro studies support the potential for e-cigarettes to cause cellular stress in vivo; however, there have been no studies to address whether exposure to e-liquid aerosols can induce cell transformation, a process strongly associated with pre-malignancy. We examined whether weekly exposure of human bronchial epithelial cell (HBEC) lines to e-cigarette aerosols would induce transformation and concomitant changes in gene expression and promoter hypermethylation. MATERIALS AND METHODS: An aerosol delivery system exposed three HBEC lines to unflavored e-liquid with 1.2% nicotine, 3 flavored products with nicotine, or the Kentucky reference cigarette once a week for 12 weeks. Colony formation in soft agar, RNA-sequencing, and the EPIC Beadchip were used to evaluate transformation, genome-wide expression and methylation changes. RESULTS: Jamestown e-liquid aerosol induced transformation of HBEC2 and HBEC26, while unflavored and Blue Pucker transformed HBEC26. Cigarette smoke aerosol transformed HBEC4 and HBEC26 at efficiencies up to 3-fold greater than e-liquids. Transformed clones exhibited extensive reprogramming of the transcriptome with common and distinct gene expression changes observed between the cigarette and e-liquids. Transformation by e-liquids induced alterations in canonical pathways implicated in lung cancer that included axonal guidance and NRF2. Gene methylation, while prominent in cigarette-induced transformed clones, also affected hundreds of genes in HBEC2 transformed by Jamestown. Many genes with altered expression or epigenetic-mediated silencing were also affected in lung tumors from smokers. CONCLUSIONS: These studies show that exposure to e-liquid aerosols can induce a pre-malignant phenotype in lung epithelial cells. While the Food and Drug Administration banned the sale of flavored cartridge-based electric cigarettes, consumers switched to using flavored products through other devices. Our findings clearly support expanding studies to evaluate transformation potency for the major categories of e-liquid flavors to better inform risk from these complex mixtures.
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Sistemas Eletrônicos de Liberação de Nicotina , Neoplasias Pulmonares , Produtos do Tabaco , Humanos , Adolescente , Nicotina/metabolismo , Neoplasias Pulmonares/patologia , Aerossóis e Gotículas Respiratórios , Células Epiteliais , Transformação Celular Neoplásica/patologiaRESUMO
Epidemiology studies link cigarillos and shisha tobacco (delivered through a hookah waterpipe) to increased risk for cardiopulmonary diseases. Here we performed a comparative chemical constituent analysis between 3 cigarettes, 3 cigarillos, and 8 shisha tobacco products. The potency for genotoxicity and oxidative stress of each product's generated total particulate matter (TPM) was also assessed using immortalized oral, lung, and cardiac cell lines to represent target tissues. Levels of the carcinogenic carbonyl formaldehyde were 32- to 95-fold greater, while acrolein was similar across the shisha aerosols generated by charcoal heating compared to cigarettes and cigarillos. Electric-mediated aerosol generation dramatically increased acrolein to levels exceeding those in cigarettes and cigarillos by up to 43-fold. Equivalent cytotoxic-mediated cell death and dose response for genotoxicity through induction of mutagenicity and DNA strand breaks was seen between cigarettes and cigarillos, while minimal to no effect was observed with shisha tobacco products. In contrast, increased potency of TPM from cigarillos compared to cigarettes for inducing oxidative stress via reactive oxygen radicals and lipid peroxidation across cell lines was evident, while positivity was seen for shisha tobacco products albeit at much lower levels. Together, these studies provide new insight into the potential harmful effects of cigarillos for causing tobacco-associated diseases. The high level of carbonyls in shisha products, that in turn is impacted by the heating mechanism, reside largely in the gas phase which will distribute throughout the respiratory tract and systemic circulation to likely increase genotoxic stress.
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Cachimbos de Água , Produtos do Tabaco , Dano ao DNA , Mutagênicos/toxicidade , Fumaça/efeitos adversos , Nicotiana/toxicidade , Produtos do Tabaco/toxicidadeRESUMO
INTRODUCTION: The efficacy of chemotherapeutic agents in killing cancer cells is mainly attributed to the induction of apoptosis. However, the tremendous efforts on enhancing apoptosis-related mechanisms have only moderately improved lung cancer chemotherapy, suggesting that other cell death mechanisms such as necroptosis could be involved. In this study, we investigated the role of the necroptosis pathway in the responsiveness of nonsmall cell lung cancer (NSCLC) to chemotherapy. METHODS: In vitro cell culture and in vivo xenograft tumor therapy models and clinical sample studies are combined in studying the role of necroptosis in chemotherapy and mechanism of necroptosis suppression involving RIP3 expression regulation. RESULTS: While chemotherapeutic drugs were able to induce necroptotic cell death, this pathway was suppressed in lung cancer cells at least partly through downregulation of RIP3 expression. Ectopic RIP3 expression significantly sensitized lung cancer cells to the cytotoxicity of anticancer drugs such as cisplatin, etoposide, vincristine, and adriamycin. In addition, RIP3 suppression was associated with RIP3 promoter methylation, and demethylation partly restored RIP3 expression and increased chemotherapeutic-induced necroptotic cell death. In a xenograft tumor therapy model, ectopic RIP3 expression significantly sensitized anticancer activity of cisplatin in vivo. Furthermore, lower RIP3 expression was associated with worse chemotherapy response in NSCLC patients. CONCLUSION: Our results indicate that the necroptosis pathway is suppressed in lung cancer through RIP3 promoter methylation, and reactivating this pathway should be exploited for improving lung cancer chemotherapy.
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Inactivation of the p16(INK4a) tumor suppressor gene and O(6)-methylguanine-DNA methyltransferase (MGMT) DNA repair gene by aberrant promoter methylation appears to be an important step in respiratory carcinogenesis after exposure to tobacco smoke and radon progeny. The determinants of aberrant promoter methylation are not well characterized. Polymorphic variants of genes of which the products are involved in pathways that modulate and repair DNA damage after carcinogen exposure may affect the occurrence of de novo promoter methylation. On the basis of their associations with risk of lung cancer, we hypothesized that functional polymorphic variants of the NADPH quinone oxidoreductase, glutathione S-transferases P1 and M1, myeloperoxidase, and XRCC1 genes are associated with p16 and/or MGMT promoter methylation in sputum from cancer-free subjects at high risk for developing lung cancer. This hypothesis was tested by conducting a cross-sectional study of 70 former uranium miners from the Uranium Epidemiological Study cohort who were at high risk for lung cancer. The polymorphic variant genotypes were characterized through PCR-RFLP on DNA isolated from peripheral lymphocytes, and the methylation status of the p16 and MGMT promoters was determined by methylation-specific PCR on DNA isolated from sputum. Subjects who had at least one GSTP1 polymorphic allele (A-to-G at bp 104) had an increased risk for MGMT methylation [odds ratio (OR), 4.8; 95% confidence interval (CI), 1.2-18.6] or for either p16 or MGMT methylation (OR, 4.4; 95% CI, 1.3-14.2). Lack of a wild-type NADPH quinone oxidoreductase allele (C at bp 609) was also associated with methylation of either p16 or MGMT (OR, 3.1; 95% CI, 1.0-9.2). These results provide the first link between germ-line functional deficits in pathways that protect the cell from tobacco- and radon-induced DNA damage, and the development of aberrant promoter methylation of the p16 and MGMT genes in the respiratory epithelium of individuals at high risk for lung cancer.
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Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Glutationa Transferase/genética , Isoenzimas/genética , Neoplasias Pulmonares/genética , NAD(P)H Desidrogenase (Quinona)/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Doenças Profissionais/genética , Escarro/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Genes p16 , Glutationa S-Transferase pi , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/etiologia , Mineração , NAD(P)H Desidrogenase (Quinona)/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Doenças Profissionais/enzimologia , Doenças Profissionais/etiologia , Polimorfismo Genético , Regiões Promotoras Genéticas , Escarro/enzimologia , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
One promising approach for early detection of lung cancer is by monitoring gene promoter hypermethylation events in sputum. Epidemiologic studies suggest that dietary fruits and vegetables and the micronutrients they contain may reduce risk of lung cancer. In this study, we evaluated whether diet and multivitamin use influenced the prevalence of gene promoter methylation in cells exfoliated from the aerodigestive tract of current and former smokers. Members (N = 1,101) of the Lovelace Smokers Cohort completed the Harvard Food Frequency Questionnaire and provided a sputum sample that was assessed for promoter methylation of eight genes commonly silenced in lung cancer and associated with risk for this disease. Methylation status was categorized as low (fewer than two genes methylated) or high (two or more genes methylated). Logistic regression models were used to identify associations between methylation status and 21 dietary variables hypothesized to affect the acquisition of gene methylation. Significant protection against methylation was observed for leafy green vegetables [odds ratio (OR) = 0.83 per 12 monthly servings; 95% confidence interval (95% CI), 0.74-0.93] and folate (OR, 0.84 per 750 microg/d; 95% CI, 0.72-0.99). Protection against gene methylation was also seen with current use of multivitamins (OR, 0.57; 95% CI, 0.40-0.83). This is the first cohort-based study to identify dietary factors associated with reduced promoter methylation in cells exfoliated from the airway epithelium of smokers. Novel interventions to prevent lung cancer should be developed based on the ability of diet and dietary supplements to affect reprogramming of the epigenome.
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Metilação de DNA , Dieta , Ácido Fólico/administração & dosagem , Fumar/genética , Escarro/fisiologia , Vitaminas/administração & dosagem , Adulto , Idoso , DNA/genética , DNA/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Fumar/metabolismo , Fumar/patologia , Escarro/química , VerdurasRESUMO
Chromosomal aberrations associated with lung cancer are frequently observed in the long arm of chromosome 6. A candidate susceptibility locus at 6q23-25 for lung cancer was recently identified; however, no tumor suppressor genes inactivated by mutation have been identified in this locus. Genetic, epigenetic, gene expression, and in silico screening approaches were used to select 43 genes located in 6q12-27 for characterization of methylation status. Twelve (28%) genes were methylated in at least one lung cancer cell line, and methylation of 8 genes was specific to lung cancer cell lines. Five of the 8 genes with the highest prevalence for methylation in cell lines (TCF21, SYNE1, AKAP12, IL20RA, and ACAT2) were examined in primary lung adenocarcinoma samples from smokers (n = 100) and never smokers (n = 75). The prevalence for methylation of these genes was 81%, 50%, 39%, 26%, and 14%, respectively, and did not differ by smoking status or age at diagnosis. Transcription of SYNE1, AKAP12, and IL20RA was completely silenced by hypermethylation and could be restored after treatment with 5-aza-2-deoxycytidine. Significant associations were found between methylation of SYNE1 and TCF21, SYNE1 and AKAP12, and AKAP12 and IL20RA, indicating a coordinated inactivation of these genes in tumors. A higher prevalence for methylation of these genes was not associated with early-onset lung cancer cases, most likely precluding their involvement in familial susceptibility to this disease. Together, our results indicate that frequent inactivation of multiple candidate tumor suppressor genes within chromosome 6q likely contributes to development of sporadic lung cancer.
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Adenocarcinoma/genética , Cromossomos Humanos Par 6 , Metilação de DNA , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Ilhas de CpG , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Predisposição Genética para Doença , Humanos , Perda de Heterozigosidade , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas , Fumar/genéticaRESUMO
Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could affect strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break (DSB) repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of DSB repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as three or more of eight genes methylated in sputum, compared with smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-PKc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with seven or more risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ataxia-telangiectasia mutated was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify DSB DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, which is, in turn, associated with elevated risk for lung cancer.