| Lung cancer is one of the most common form of malignant diseases and the leading cause of cancer-related mortality worldwide. China is a most country of tobacco production and consumer in the world. Recently, with the acceleration of industrialization, the problem of air pollution in our country is much more serious than before. According to recent data, lung cancer is the leading cause of cancer incidence in our country, which does great harm to urban and rural population.Tobacco smoke is a main contributory factor in the development of lung cancer. The incidence of lung cancer in smokers is 10 times more than nonsmokers. Smoking cessation can reduce the incidence of lung cancer. It is reported that tobacco smoke contains thousands of chemicals including oxidative gases, cyanide, heavy metals, and more than 69 carcinogens. The most significant component of these carcinogens is polycyclic aromatic hydrocarbons and nicotine metabolites such as benzopyrene and N-nitrosonornicotine (NNN). The harms of passive smoking should also be noted. It is reported that inhaled sidestream smoke, the main component of secondhand smoke, involving more than 250 carcinogens, which is four times more toxic than mainstream smoke. There is evidence that long term exposure to tobacco may trigger cellular mechanisms implicated in DNA damage response (DDR). Smoking is deeply involved in a great range of biological activities and disease states, such as cancer, cardiovascular diseases, and neurodegeneration.DNA repair plays a fundamental role in the maintenance of genomic stability against the threat from exogenous and endogenous stress, avoiding the accumulation of DNA damage, and its detrimental consequences on chromosomal abnormalities, sensitivity to genotoxins, and cell function. Maintenance of genomic stability is critical for the wellbeing of organisms. The cells may start the apoptosis process if they failed to repair damage DNA. The abnormal DNA damage repair is a risk factor for cancer. Tobacco smoking is the leading cause of lung cancer, while the role of cigarette smoke-induced DNA damage repair in lung cancer is largely unknown.In order to explore those issues, we performed the present study to investigate cigarette smoking-induced DDR and repair in airway epithelial cells.Part I Cigarette smoking-induced DNA damage response in airway epithelial cellsObjective:To study whether cigarette smoking-induced DNA damage response (DDR) in airway epithelial cells, as well as underlining mechanisms.Methods:Cigarette smoke-induced human airway epithelial cells proliferation and apoptosis were assessed by flow cytometry, colony formation assay, and CCK8 assay. Senescence-associated β-galactosidase and chromosome structure were also observed. DDR was detected by a DNA damage marker, yH2AX (immunofluorescence, IF) and cell cycle arrest of HBE cells after treated with CSE were observed. The DNA fiber assay was performed to study DNA replication when airway epithelial cells were exposed to CSE. After treated with CSE, the expression of XRCC1, RPA, USP1, FANCD2, and FANCI was analyzed by Western Blot. FANCD2 and FANCI mRNA was investigated by Q-PCR.Results:CSE can inhibit the proliferation of HBE cells and promote apoptosis in a time- and dose-dependent manner. CSE treatment induced a significant increased in fragile site expression and DDR of HBE cells. No significant in cell aging was observed between CSE treatment group and control group. After CSE addition, the majority of yH2AX foci were observed in cells also expressing Cyclin A. DNA replication in CSE and control group was 0.2kb/min-0.8kb/min and 0.6kb/min-1.2kb/min, respectively. Moreover, CSE can downregulation the expression of FANCD2 and FANCI.Conclusion:Cigarette smoking-induced DDR in airway epithelial cells by downregulation of FANCD2 and FANCI.Part II IL-17 promotes cigarette smoke-induced genomic instability and DNA damage responseObjective:To investigate whether cigarette smoke was able to induce DNA damage response (DDR) and genomic instability in multiple tissues as well as whether interleukin (IL)-17 involved in cigarette smoke-induced genomic instability.Methods:C57BL/6 and IL-17-/- mice were randomly assigned to different treatments (air or cigarette smoke) at the time of purchase to minimize any potential bias. Mice were exposed to smoke for 12 weeks and DDR was analysis in multiple tissues involving brain, esophagus, lung, heart, liver, stomach, intestine, spleen, kidney, and bladder. Hematoxylin and eosin (HE) staining was performed to study pathological forms of multiple tissues. To see whether IL-17 can trigger DNA damage, we investigated DDR of airway epithelial cells by using immunofluorescent staining and Western Blot. The inhibitors of ATM and ATR were used to study whether they can prevent IL-17-induced phosphorylation of H2AX. IL-17 expression in the lung and bladder and associated with DDR was investigated in mice and human samples.Results:The current study demonstrated that cigarette smoke-exposure induced DDR in the lung and bladder, but not for brain, esophagus, heart, liver, stomach, intestine, spleen, or kidney. In addition, cigarette smoke-exposure promoted IL-17 expression and airway inflammation. A positive correlation was observed between IL-17 expression and DDR. It is surprising that DDR in the lung of mice was diminished in IL-17-/- mice when exposed to cigarette smoke. In vitro, IL-17 treatment induced DDR of HBE cells. The inhibitors of ATM and ATR can reduce IL-17-induced phosphorylation of H2AX. IL-17 and DDR was up-regulation in the lung and hladder of smokers than that of nonsmokers. In addition, a positive association was found between IL-17 and DDR in the lung and bladder of human samples. Conclusion:Cigarette smoke-induced genomic instability in the lung and bladder. IL-17 promotes cigarette smoke-induced genomic instability and DDR.Part III Smoking-promoted oxidative DNA damage response is highly correlated to lung carcinogenesisObjective:To investigate oxidative DNA damage response in smoking and nonsmoking patients with lung cancer.Methods:Cigarette smoke-induced human airway epithelial cells oxidative DNA damage response were assessed by immunofluorescent. C57BL/6 mice were randomly assigned to different treatments (air or cigarette smoke) at the time of purchase to minimize any potential bias. Mice were exposed to smoke for 12 weeks and 24 weeks and oxidative DNA damage response of lung tissues was assessed (IHC,8-OHdG).88 patients with suspected lung cancer and undergo bronchoscopy were enrolled in this study. Clinical information regarding patient characteristics was based on patient records and registries. All patients had histological confirmed. There were 50 lung cancer patients and 38 patients with benign disease. There was no statistically significant difference in age, sex distribution, or smoking between lung cancer patients and benign disease group. The levels of 8-OHdG in smokers and nonsmokers of lung cancer patients were studied.Results:We observed a higher 8-OHdG expression and secretion in airways of lung cancer patients than that of noncancer controls.8-OHdG expression was associated with the TNM stage. Additionally, cigarette smoke-induced oxidative DNA damage response was observed in bronchial epithelial cells in vitro and in vivo study. A statistical significance correlation was found between the levels of 8-OHdG and smoking index. With a cut-off value of 2.86ng/ml,8-OHdG showed a sensitivity and specificity of 70.0%and 73.7%, respectively, to identify a patient with lung cancer.Conclusion:Cigarette smoke was associated with oxidative DNA damage response. In addition,8-OHdG can serve as a useful biomarker for lung cancer. |
