The Pathogenesis Research Of Race Elements,Antioxidant Enzymes And Differential Proteomics In Early Laryngeal Squamous Cell Carcinoma Related To Smoking

Laryngeal is the most prevalent cancer of head-and-neck cancer, with a increasing ratio year by year. Glottic larynx carcinoma is the most common cancer and makes up to half of laryngeal. Early glottic cancer will change the vibratory movements of the vocal cords with a higher cure rate after early diagnosis. Supraglottic cancer and subglottic cancer are the additional forms of laryngeal. Because both of these cancers have no obvious symptoms at early stage and lymphoid tissue is abundant, their prognosis are poor. In clinic, laryngeal can be treated with surgery in addtion to radiotherapy. If local invasion and cervial metastasis appear after treatment, they will seriously affect patients'prognosis. Therefore, early detection, early diagnosis, early treatment and close inspection after surgery are the keys to improve the survival rates of patients and postperative quality of life.Epidemiological evidence has established that smoking is an important enviromental factor for laryngeal carcinogenesis. Death from laryngeal cancer is20times more likely to occur for heavy smokers than for nonsmokers. The relative risk was evidently increased for SCCs after its histological types were analyzed. Smoking can induce oxidative stress of critical physiological structures thus have a negative effect on tissues. Consecutive intracellular reduction of molecular oxygen can generate reactive oxygen species (ROS) including superoxide anion radical (O2-), hydrogen peroxide (H2O2) and hydroxy radical (·OH). Under normal physiological conditions, cells have the ability to defend themselves against ROS by way of their own antioxidant mechanisms, like enzymes and trace elements and vitamins. SOD, CAT, GSH-Px are the most effective enzymes. O2-can be regarded as the principle ROS and is disposed into H2O2and O2by SOD. H2O2is regarded as the central ROS in carcinogenesis and disposed by CAT and GSH-Px into oxygen and water.·OH are highly reactive, but short-lived molecules that trigger DNA damage and give rise to mutations.·OH can also react with other cellular molecules, thereby denaturing enzymes or strucural proteins o initiating peroxidation of polyunsaturated fatty acids. Trace elements play important roles in a number of biological processes. They activate or inhibit enzymatic reactions by competing with other elements and metalloproteins for binding sites. They can also modulate cell membrane permeability, regulate gene expression, and participate in electron transport as well as in the synthesis of hormones and vitamins. Any variation in one of these systems may cause imbalance and cellular damage, ultimately resulting in malignant transformation. Then abnormal hematological markers indicate the possibility occurance of early laryngeal.Some studies have reported proteins relevant to laryngeal carcinoma. A systematic review of mucins on the protective role on the larynx showed MUC1is definitely expressed in laryngeal squamous cell carcinoma (SCC). Over-expression of CD44could be used as a prognostic factor in laryngeal squamous cell carcinoma. Calponin may be a prognostic factor of laryngeal myoepithelial carcinoma. Other proteins are also related to the progress of laryngeal carcinoma, such as REPP86, DJ-1, SATB1, CRBP-1, fascin and Cap-43. However, little studies were performed on a systematic protein or gene level to explore the roles of these proteins in different laryngeal carcinoma progress. Therefore, the study of proteome of serum proteins of laryngeal will provide new insight into the pathomechanisms, prognosis and progess of laryngeal. The study determined the antioxidant enzymatic activities and trace element levels in tobacco smoke T1glottic larynx carcinoma patients as well as in smokers and nonsmoker healthy controls for the first time. At the same time, a comparative proteomic analysis were performed, with two dimensional gel electrophoresis followed by mass spectrometry and database searching, the proteins which were up or down regulated in patients with laryngeal carcinoma. The results maybe helpful for early diagnosis, discovery of abnormal hematological markers, and prognosis. Objective:To determine the variations in the plasma antioxidant enzymatic activities and trace element levels in early stage laryngeal carcinoma and to better evaluate the serum protein alterations in patients with laryngeal carcinoma during surgical treatment process in order to find target proteins for clinical diagnosis, treatment and prognosis.Methods:SOD, CAT, and GSH-Px activities were determined by ELISA. Cu, Zn, Pb, Ca, Mg, Fe, and Cd were measued using atomic absorption spectrophotometry (AAS). Serum was extracted from patients with laryngeal carcinoma between pre-and post-operation group. A comparative proteomic analysis was performed using two-dimensional gel electrophoresis, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry and database searching. ELISA kits were used to detection the MDA and SOD levels in different groups.Results:A total of45tobacco smoke laryngeal carcinoma patients (group A) and80healthy volunteers (group B,40smokers; group C,40non-smokers) were examined in part Ⅰ. SOD and GSH-Px activities were not significantly higher in group A than those in group B, but were significantly lower than those in group C. The highest levels of Mg and Fe were found in group A and significant difference was found between groups A and C as well as between groups B and C Significant differences were found to exist between the levels of Cd in groups A and C as well as those in groups A and B, with the highest level found in group A. The highest concentration of Cu was in group C, with a significant difference between groups A and C as well as between groups B and C. In part Ⅱ, statistical analysis indicated that16gel spots corresponding to12proteins altered their expression significantly between the two groups of patients with laryngeal carcinoma. Of these12proteins,6proteins were up-regulated in the pre-operation group. The subsequent Western Blot confirmed the results of proteome analysis. MDA levels is higher than control group and post-operation group (P<0.01). SOD levels is lower in the pre-operation group than control group (P<0.05) with no significant meaning compare to that in the post-operation group (P>0.05).Conclusion:The results indicate that tobacco use lowers the levels of antioxidant enzymes. In addition, abnormal levels of trace elements may play important roles in cancer progression. In addition, differential proteomics analysis indicated the potential roles of the oxidant stress and immune response as target in monitoring and treating laryngeal carcinoma and may be helpful for pathological progress of laryngeal. PART ONE:PRELIMINARY PROTEOMIC ANALYSIS OF HUMAN SERUM FROM PATIENTS WITH T1GLOTTIC LARYNGEAL CARCINOMAObjective:Different serum protein levels in pre-and post-operative T1glottic laryngeal carcinoma patients were analyzed using proteomics methods to determine the tumor biomarker proteins.Methods:Comparative proteomic analysis of human sera between pre-and po st-operative T1glottic laryngeal carcinoma patients was performed using two-dimensional (2D) gel electrophoresis, in combination with matrix-as si ste d laser desorption ionization time of flight mass spectrometry and database searching, to determine alternative proteins in T1glottic laryngeal carcinoma patients. Western blot analysis, as well as the measurement of mal ondialdehy de (MDA) and superoxide dismutase (SOD) levels, was also performed.Results:Image analysis of2D gels indicated that16protein spots corresponding to12proteins exhibited significant different expression levels between the two groups of T1glottic laryngeal carcinoma patients. Six protein expression levels were upregulated. Western blot analysis confirmed the results of proteome analysis. MDA levels in the pre-treatment group were higher than those in the control and post-treatment groups. By contrast, SOD levels in the pre-treatment group were lower than those in the control and post-treatment groups. Conclusion:Proteomics results combined with bioinformatics analysis indicate the important roles of oxidant stress and immune response as targets in the monitoring, as well as treatment, of T1glottic laryngeal carcinoma. The differentially expressed proteins may also reflect the pathological progress of T1glottic laryngeal carcinoma and should be studied further. PART TWO:ANTIOXIDANT ENZYMATIC ACTIVITIES AND TRACE ELEMENTS IN TOBACCO SMOKE T1GLOTTIC SQUAMOUS CELL LARYNX CARCINOMAObjective:The variations in the plasma antioxidant enzymatic activities and trace element levels in early stage laryngeal carcinoma were determined.Methods:We investigated the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, as well as the following trace elements:copper (Cu), zinc (Zn), lead (Pb), calcium (Ca), magnesium (Mg), iron (Fe), and cadmium (Cd), in tobacco smoke T1glottic larynx carcinoma.Results:A total of45tobacco smoke laryngeal carcinoma patients (group A) and80healthy volunteers (group B,40smokers; group C,40non-smokers) were examined. SOD and GSH-Px activities were not significantly higher in group A than those in group B, but were significantly lower than those in group C. The highest levels of Mg and Fe were found in group A and significant difference was found between groups A and C as well as between groups B and C. Significant differences were found to exist between the levels of Cd in groups A and C as well as those in groups A and B, with the highest level found in group A. The highest concentration of Cu was in group C, with a significant difference between groups A and C as well as between groups B and C. Conclusion:The results indicate that tobacco use lowers the levels of antioxidant enzymes. In addition, abnormal levels of trace elements may play important roles in cancer progression. The combined determination of antioxidant enzyme activities and of trace elements levels may be of value for the early diagnosis of laryngeal carcinoma.