The Correlation Of Blood Flow Classification With Angiogenesis Markers In Lung Adenocarcinoma And The Effects Of Low-Dose Cadmium On VEGF Expression In Lung Adenocarcinoma Cells

Nearly half a century, in China and even around the world, the morbidity and mortality of lung cancer has been on the rise. The most common types of lung cancer include large cell carcinoma, squamous cell carcinoma, adenocarcinoma and small cell lung cancer. The first three of them are called non small cell lung cancer. Previous research data showed that pathological predominant in male lung cancer in type is squamous cell carcinoma. But in recent years, due to the low tar content of tobacco products, lung cancer incidence has more than squamous cell carcinoma, which has become the main type of lung cancer. And women’s susceptibility to lung adenocarcinoma is increasing. Early diagnosis is an important means to reduce the mortality rate of all malignant tumors, and it is no exception for lung cancer. Because of the early stage of lung adenocarcinoma, there is no obvious clinical symptoms, so it is very difficult to diagnose in early stage. Tumor early lymph node and distant metastasis is one of the important factors affecting the prognosis of lung adenocarcinoma. Most lung cancer patients have been in advanced stages of the disease in the diagnosis, even had regional lymph node metastasis and distant metastasis. Although with high speed current molecular biology techniques and clinical skills development, our understanding of the molecular biology mechanism of lung cancer has made some progress, but we still have insufficient understanding of its pathogenesis. There is still a lack of effective early diagnosis of adenocarcinoma of the lung and the criteria for judging the prognosis. Therefore, more studies are needed to elucidate the molecular mechanisms underlying the pathogenesis of the disease. By which, we look forword to find a more appropriate early diagnosis, treatment and prevention of lung adenocarcinoma metastasis effective method, furthermore, to prolong survival time, improve the life quality of patients.The biological characteristic of malignant tumor is unlimited growth. On the one hand, it will take a lot of nutrients needed for normal tissue growth, which causes the body to feel weak., gradually emaciated, even cachexia. On the other hand, the proliferation of cancer cells also produces a lot of toxins, which can cause the body to produce a variety of symptoms, such as persistent low fever, etc. Because of the invasion and metastasis of malignant tumor, it is especially easy to transfer and recurrence. Tumor cells can be transferred to the surrounding tissues or organs or distant organs by direct transfer, blood route and lymphatic pathway, form new metastatic foci. So the prognosis of malignant tumor is always unsatisfactory. Tumor angiogenesis is considered as a necessary condition for the occurrence of tumor invasion and distant metastasis, because the new vessels could provide the necessary growth of sufficient oxygen and nutrients for tumor. Some studies have showed that HIF1-a is a transcription factor with the activity of specific hypoxia, whose expression is very wide, is involved in tumor angiogenesis in many malignant tumors. The extracellular matrix has the function of preventing the cell from moving freely, is regarded as a barrier to prevent cell movement. If it is degraded, it loses the function of preventing cell movement, which is beneficial to the occurrence and metastasis of tumor cells. Matrix metalloproteinases can degrade various types of collagen in extracellular matrix, matrix damage an important barrier to prevent tumor cell invasion, thereby promoting tumor cell invasion or metastasis. Metalloproteinase -9 is a kind of matrix metalloproteinases, which plays an important role in the invasion of tumor cells. It has been reported that there is a correlation between the prognosis of Metalloproteinase -9 expression and non-small cell lung cancer.In the past, it was believed that the application of ultrasound in the lung was greatly restricted because of the strong reflection of the gas in the lungs and the interference of the shoulder blades and ribs. But in recent years, with the improvement of the ultrasonic instrument and the progress of technology, ultrasound in the chest wall, peripheral lesions of the lung, pulmonary edema and other diseases is in the gradual increase in the number of applications. Ultrasound for lung cancer, mainly adjacent to the chest wall of peripheral lung cancer, lung cancer adjacent to the bottom of the brittle muscle and the combination of obstructive pneumonia or some of the deep lung cancer diagnosis has its unique characteristics. Combined with CDFI examination of tumor blood supply, and thus provide more data to distinguish between benign and malignant tumors.The first part of this study is to detect the expression of MMP9 and HIF1a in lung tumor tissues, with ultrasound showed that the blood supply of tumor, discuss the application of ultrasonic blood flow classification in evaluating the degree of angiogenesis in peripheral lung cancer, provide a theoretical basis for the non-invasive diagnosis of peripheral lung cancer and the effect of the treatment of lung cancer.Comprehensive various theories of current medical research, the most current academic thought that the malignant tumor is a variety of factors, long-term effects of interaction between the results. In general, many factors that lead to malignant tumors include internal and external factors. Genetic and gene mutations and other genetic factors, endocrine factors and immune factors belong to the internal factors of the body, chemical factors, physical factors, biological factors and so on are all external environmental factors. Now with the rapid development of economic construction in our country, most of the chemical pollutants and heavy metals and other pollutants will with the application of pesticides, chemical waste emissions, metal smelting waste residue dumping and other various industrial activities into our living environment. Environmental pollution has become an important factor in the occurrence of malignant tumors. There have been a number of reports, some of our country’s city due to the living environment is some chemical product or some kind of heavy metal pollution, resulting in malignant tumor of the life of the masses in the region concentrated phenomenon, and even the emergence of "cancer villages". Environmental pollution as a cause of malignant tumor can not be ignored.Some studies have found that the heavy metal pollutants play a more important role in the process of tumor. On the one hand, the heavy metal pollutants are absorbed into the body can induce mutations in the gene, thus further the possibility of malignant tumor, on the other hand, the body of the gene mutation, which is more sensitive to the stimulation of heavy metals in the surrounding environment. When both exist at the same time in the same body, the possibility of the occurrence of tumors will be greatly increased. At present, the main source of cadmium pollution in human living environment is the modern industrial production. Heavy metal cadmium has been widely used in paint, electroplating and other industries. Cadmium is very serious harm to human health. Diet, smoking and occupational exposure are the main sources of chronic exposure to trace cadmium pollution in human beings. Heavy metal cadmium has been listed as one of the toxic substances in the United States. As early as 1993, cadmium was classified as a carcinogen by the International Institute for cancer research. Cadmium in the body of biological half-life is very long, up to 10～30 years, therefore, cadmium in the body is very easy to accumulate. Long term low dose exposure to cadmium may have a lot of serious harm to the human body. Although the relationship between cadmium exposure and some of the organs of the tumor is not clear, but it has been confirmed that cadmium in the human body can cause the occurrence of lung cancer. There have been many studies on the mechanism of cadmium induced tumor in cell experiments and rodent experiments, and some experiments have found that there is a dose dependent relationship between cadmium and lung adenocarcinoma in rodents. But cadmium is how to promote the occurrence of lung adenocarcinoma, the specific mechanism is not clear.Angiogenesis refers to the process of creating new blood vessels in the presence of blood vessels. The dense network of blood vessels in the body provides the body with the oxygen and nutrients needed to be exchanged, and the metabolic products. It also plays a very important role in the growth of solid tumors, such as the proliferation and metastasis of tumor. According to reports, in an environment with low oxygen content, tumor cells can secrete vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and other factors to promote angiogenesis factor. There are many kinds of growth factor receptors on the vascular endothelial cells, such as VEGF receptor 2, FGF receptor 1 and so on. These receptors can promote the proliferation and migration of vascular endothelial cells in the body after the combination of these receptors and their corresponding growth factors. At present, the vascular endothelium is one of the targets of the toxic effects of cadmium. But so far, there is still a controversy about the effect of cadmium on the growth of tumor blood vessels. Studies have shown that in a certain concentration range, cadmium can increase the activity of VEGFR2 and promote the formation of vascular endothelial tissue. However, studies have also indicated that cadmium can damage the ability of human breast cancer cells to induce angiogenesis. Therefore, the effect of cadmium on angiogenesis in lung adenocarcinoma is not yet clear.For cadmium in lung adenocarcinoma angiogenesis to elucidate the role and its mechanism, we observed effect on human lung adenocarcinoma cells to vascular endothelial growth factor (VEGF), a fibroblast growth factor (bFGF) expression and secretion of cadmium, we observed the effects of cadmium treatment on the proliferation, migration and transfer of HUVECs in A549 tumor conditioned medium and HUVECs co culture, we observed the effects of cadmium on the proliferation, migration and apoptosis of A549 cells, in order to find the role and mechanism of cadmium in angiogenesis, malignant tumor formation, invasion and metastasis of lung adenocarcinoma.Part one:The correlation of blood flow classification with angiogenesis marks in lung adenocarcinomaObjective:To evaluate the blood supply of peripheral lung adenocarcinoma by ultrasound, and to detect the distribution density of the tumor tissue, and to evaluate the blood supply of the tumor. At the same time, we detected the expression level of Hypoxia inducible factor-la (HIF-1α) and MMP9 in lung adenocarcinoma tissue, and compared the relationship between the peripheral lung adenocarcinoma angiogenesis factor and the corresponding clinical parameters.Research methods:The 66 cases by postoperative pathological diagnosis for lung adenocarcinoma of peripheral lung cancer patients before operation of conventional two-dimensional ultrasonography and color Doppler ultrasonography. According to the Doppler ultrasound shows blood supply richness is divided into 0-Ⅲ grades, which 0-Ⅰ degree for lack of blood supply group, grade Ⅱ～Ⅲ for the rich blood for group. We used the tumor tissue of patients with surgical resection, by immunohistochemistry method detect the expression of HIF-la and MMP9 in lung cancer tissues.Results:A total of 66 patients of lung adenocarcinoma were examined. By CDFI blood flow classification,16 patients were identified as Class 0. Among them,6 cases were HIF-la positive (positive rate:37.5%), and 5 cases were MMP9 positive (positive rate:31.2%).11 patients were identified as Class I. Among them,6 cases were HIF-la positive (positive rate:54.5%), and 5 cases were MMP9 positive (positive rate:45.4%).23 patients were identified as Class II. Among them,16 cases were HIF-la positive (positive rate:69.5%), and 15 cases were MMP9 positive (positive rate:65.2%).16 patients were identified as Class III. Among them,12 cases were HIF-la positive (positive rate:75%), and 13 cases were MMP9 positive (positive rate:81.2%).The expression of HIF-1α and MMP9 was positively correlated with the blood flow classification in the lung adenocarcinoma.Conclusion:The blood flow classification has correlation with angiogenesis marks in lung adenocarcinoma. This has certain significance in diagnosis and treatment of lung cancer clinical selection scheme.Part two:Study on the mechanism of angiogenesis in lung adenocarcinoma induced by low concentration cadmiumObjective:To investigate the influence of expression of VEGF mRNA and b-FGF mRNA in A549 cells treated by Cd, To investigate the influence of expression and secretion of VEGF in A549 cell treated by Cd, and research the effects of HUVECs cell proliferation,apoptosis and migration treated by Cd-A549 CM.Materials and Methods:1. The influence of the expression of VEGF and b-FGF in A540 cells treated by CdA549 cells were treated by Cd at the concentration of 0,0.1,0.5,1,5, and 10μM. Thereafter, the level of VEGF mRNA and b-FGF mRNA were analyzed by Quantitative Real-Time PCR, the proteins of VEGF were detected by Western blotting, the proteins secretion level of VEGF and b-FGF were detected by ELISA.2. The influence of cell proliferation on HUVECS treated by Cd-A549 cell CM.Make Cd-A549 cell conditioned medium.HUVECs cells were treated by A549 CM or Cd-A549 CM respectively. Thereafter, the proliferation of HUVECs cells was assessed by MTT method and ECIS and the apoptosis rate was analyzed by flow cytometry.3. The influence of the expression of HIF-1α in A540 cells treated by Cd A549 cells were treated by Cd at the concentration of 1μM. Thereafter, the proteins level of HIF-1α were detected by Western blotting.4. The influence of cell proliferation, apoptosis rate on A549 cell treated by Cd..Result:1. Being treated singly by 0.5μM、1μM Cd,the expression level of VEGF mRNA was significantly increased comparing with the control group (0.5μM:1.52±0.17, p < 0.05; 1μM:2.5±0.39, p<0.01), but this does not occur in the other groups which were treated by O.1μM、5μM、10μM Cd (0.1μM:1.07±0.04,p= 0.05; 5μM:1.53 ±0.51,p=0.154; 10μM:1.11±0.23,p=0.468). After being treated with Cd, there were no marked change of b-FGF mRNA expression in A549 cells compared with the control group(p>0.05).2. Being treated with 1μM Cd could obviously up-regulate the level of VEGF protein in A549 cells (Control:1.11±0.13; Cd:1.79±0.17,p< 0.05). Level of VEGF protein secretion were obviously up-regulated in A549 after being treated with 1μM Cd. After being treated with Cd, there were no marked change of b-FGF protein seretion in A549 cells compared with the control group(p>0.05).3. Being treated with Cd-A549 CM could obviously promote the growth of HUVECs (1.25±0.06, p< 0.01), The trans-endothelial resistance of HUVECs was significantly increased in Cd-A549 CM (1.64±0.19,p<0.05), The results of flow cytometry showed no significant changes in cell apoptosis.4. Being treated with 1μM Cd could obviously up-regulate the level of HIF-la protein in A549 cells (Control:1.11±0.13; Cd:1.79±0.17,p<0.05). After being treated with Cd, level of HIF-la protein seretion were obviously up-regulated in A549 cells compared with the control group.5. Low Dose of Cd has No Significant Effect on Proliferation, Migration, and Apoptosis of A549 Cells.To examine the effects of Cd on A549 cells, CdC12 at the concentrations of 0.1, 0.5,1,5,and 10μM was applied to the cell culture. MTT assay showed that 24-h Cd treatment did not significantly change A549 cells’proliferation at all concentrations. Cell migration was evaluated by a wound healing assay, and the wound closure rates were largely unchanged in all Cd-treated groups of A549 cells. In addition, apoptosis was measured with Annexin V-FITC/PIdouble-labeled flow cytometry. No significant change in the apoptotic rate was observed in Cd-treated A549 cells.Conclusions:1. Low-dose Cd could specifically upregulates the expression and secretion of VEGF., could obviously promote the proliferation and migration of HUVECs, it might facilitate the development of tumors through this pro-angiogenic effects.2. Low-dose Cd can upregulates the secretion of VEGF by promoting secretion of HIF-1α..3. Low-dose Cd does not affect proliferation, migration, or apoptosis of lung . adenocarcinoma cells.