| Lung cancer remains a serious public health problem and is the first cause of cancer death worldwide, and the overall 5-year survival rate for all stages is 14-17% for Non-small-cell lung cancer and 6% for small-cell lung cancer. Clinical and epidemiologic studies have suggested a strong association among chronic infection, inflammation, and cancer. Immune system plays a critical role in maintaining tissue homeostasis, cell turnover, tissue remodeling, and preventing infection and cell transformation. The inflammatory component in the development of the neoplasm includes a diverse leukocyte popμlation; these components are considered inflammatory tumor key factors promoting tumor progression due to its ability to release a variety of cytokines, chemokines, and cytotoxic mediators such as reactive oxygen species (ROS), metalloproteinases, interleukins, and interferons. Cancer-related inflammation affects many aspects of malignancy, including the proliferation and survival of malignant cells, angiogenesis, tumor metastasis, and tumor response to chemotherapeutic drugs and hormones.Lung cancer was the most commonly diagnosed cancer as well as the leading cause of cancer. Among female, it was the fourth most commonly diagnosed cancer and the second leading cause of cancer death. In both developed and developing countries, breast and lung cancer are the leading cause of women and men in the most common cancer and cancer deaths evil. Lung cancer was observed between the occurrence and trends in different countries, or each country can be seen in the ratio of men and women occurred very close relationship with the frequency of use of tobacco. Clinical and epidemiologic studies have suggested strong association between chronic infection, inflammation, and cancer. Up to 20% of cancers are linked to chronic infections,30% can be attributed to tobacco smoking and inhaled pollutants (such as silica and asbestos), and 35% to dietary factors (20% of cancer burden is linked to obesity).Now study found that nitric oxide synthase (NOS) has an important significance in the process of tumor genesis. First discovered nitric oxide synthase (NOS) is purified from brain tissue out, named as neuronal NOS or I-NOS (nNOS or NOS-1); subsequently discovered inducible NOS, called Ⅱ NOS (iNOS or NOS-2); and later discovered the endothelial or Type Ⅲ NOS (eNOS or NOS-3). These three nitric oxide synthase gene of different types are located in three different chromosomes. NOS-2 can be induced by lipopolysaccharide (LPS) or a number of pro-inflammatory cytokines (IL-1, IFNγ, TNFα), and rapidly expressed in vivo, produce large amounts of nitric oxide which is widely distributed in different types cells. Excessive nitric oxide promotes tumor cell proliferation significance, and in many pathological conditions will also promote production of nitric oxide, such as (1) acute tissue damage and various inflammatory diseases (2) neurological diseases (3) autoimmune diseases (4), and angiogenesis-related diseases (5) diabetes.iNOS was separated in the macrophages of mice out first. Macrophages can produce nitric oxide and reactive oxygen via inducible nitric oxide synthase, and then produce superoxide anion which can kill microorganisms. But the excess of nitric oxide, reactive oxygen and superoxide anion can increase oxidative stress and tissue damage of the body, can cause chronic inflammation of the pathological state in the body. In some chronic inflammatory diseases, macrophages are considered a risk factor to induce tumors.In mouse lung spontaneous squamous cell carcinoma model (Lori-IKKαK44A/K44A), IKKa kinase is inactivation, IKKa level is down on the occurrence of squamous cell carcinoma of the lung.This resμlt have an important role in promoting the study of lung squamous cell carcinoma. In this study, it was shown that the incidence of lung tumor was associated with tumor microenvironment and iNOS level and the expression of inflammatory cytokines and chemokines factor were increased on tumor site or proximal adjacent tissues. It is similar to the incidence of the human condition. Meanwhile, the study found that by eliminating macrophages can reduce lung tumor inflammatory cytokines significantly. In order to observe the important role of iNOS in lung squamous cell carcinoma, we generate iNOS gene knockout mice (iNOS-/-) on the basis of this animal model by genetic hybridization, in order to observe the incidence of tumors in mice and iNOS therapeutic effect on the tumor.Objective:Through iNOS knockout mice to observe the important role of iNOS signaling pathway in lung squamous cell carcinoma and explore the therapeutic implications of iNOS in human lung squamous cell carcinoma.SubjectsAll mice used in this study were cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the National Institutes of Health. All mice were divided into three groups:WT, L-IKKαK44A/K44A, L-IKKαK44A/ K44ANOS2-/- by genotype. Choose 50 mice of each group to observe.18 mice of each group were observed continuously according mice activity, eating status, shedding skin, hair losing, respiratory rate and degree of dyspnea. The mice which is looked abnormal will be euthanized and harvest lung and spleen tissues.Methods1. Screening by PCR in mice genotype required, including Loricrin gene positive, IKKaK44A; K44A and NOS2-/- mice homozygous mutation. According to the need to keep heterozygous mice for generation.2. Using TRIzol reagent to isolate RNA of tissues and cells, using Tetro cDNA Synthesis Kit (BIOLINE, USA) to reverse RNA to cDNA, and then detect RNA level of IL5, IL17α in the lung tissue of three groups, each experiment repeat 3 times. At the same time with Real-time PCR for RT2 Profiler PCR-array detection lung and CD4+ T lymphocyte cell of three groups of mice.3. By flow cytometry to sort splenic CD4+ T cells. Using 6-8 week old mice,3-5mice in each group, isolating extract the CD4+ T lymphocytes selected only by PE CD4, APC-Cy7 CD8, and 7ADD labeled spleen cells, after extraction of RNA detected cytokine levels.4. Measure the number of macrophages and lymphocytes cytometry in mice lungs by flow cytometry. Incubate lung cells, using 10-12 week old mice,3-5 mice in each group. Mononuclear cells labeled antibody:FITC CD45, PE CD11c, PE-Cy7 CD11b, APC F4/80, APC-Cy7 MHCII,700 GR1. Lymphocytes labeled antibody. FITC CD45, PE CD4, PE-Cy7 B220, APC CD3, APC-Cy7 CD8. The experiment was repeated 3 times.5. Using Western blot to detect Trim29 and cyclooxygenase-2 (COX-2) in mice lung tissue.3 mice were selected in each group which were 5 month old.The experiment was repeated 3 times. Detect iNOS and Argeanse-lin mouse macrophages.3 mice were selected in each group which were 6-8 week old. The experiment was repeated 3 times.6. Murine bone marrow transplant. Using y-ray irradiate 5 mice in each group which were 6-8 week old, and then receive bone marrow transplant. Select L-IKKaK44A/K44A and L-IKKαK44A/K44ANOS2-/- mice which were 12 week old as Donor. One week before the transplant and two weeks post-transplant, the recipient mice feeding antibiotics water. After two months of transplant the receiving mice were sacrificed to observe the incidence of lung tumors in mice.7. To observe the changes in DNA damage in lung cells. By immunofluorescence staining, using 8-OH-DG to stain lung tissue of the three groups.3 mice was selected in each group which was 6 month old.8. Statistical Methods:All experimental data were input GraphPad Prism5 statistical software, shown as mean ± SD, using t-TEST for statistical analysis, p<0.05 considered statistically significant.Resμlt1. In the continuous observation of 18 mice, WT mice can be healthy survival for 1 year as a negative control.15 of L-IKKaK44A’K44A mice had lung tumors before six month old and are the pathological examination showed lung squamous cell carcinoma.The remaining three mice in 7-10 months were all dead and pathology resμlts were squamous cell carcinoma.12 of L-IKKaK44A/ K44ANOS2-/- mice were euthanized at 6 months and only 2 of them were detected the squamous cell carcinoma in the lung. And the remaining 6 mice were observed continuously, and can survive to 12 months without lung tumors. Using histopathological sections HE staining, compare to L-IKKaK44A /K44A, the inflammatory infiltration in the lung of L-IKKaK44A;K44ANOS2-/-group was significantly reduced, By immunohistochemistry and flow cytometry, we detected the number of macrophages in the three groups. The results showed that compare to L-IKKaK44A; K44A, the MAC number in L-IKKaK44A/K44ANOS2-/- group were significantly reduced, p<0.001, there is a significant statistical significance.2. In order to confirm the role of iNOS in the bone marrow, we were used L-IKKaK44A’K44Aand L-IKKαK44A/K44A NOS2-/- mice as donor to transplant bone marrow into three groups. And to observe the tumor occurrence after two months. The resμlts show that either the donor or acceptor which had iNOS-/-, the lung cancer incidence were more reduced than iNOS+/+.It showed that not only in the epithelium cells and but also in macrophages, iNOS-/- can inhibit the tumor growth.3. It also showed that Trim29 and COX2 expression has significantly decreased in L-IKKaK44A/K44ANOS2-/- mice, indicating that in iNOS-/- can decrease the oxidative stress in vivo, and reduce cell DNA damage. At the same time, we used 8-OH-DG to do immunofluorescence staining and confirmed the result.4. Detecting some cytokines and chemokines of lung and T cells (IL5, IL17α). Compare to L-IKKaK44A’K44A, L-IKKaK44A/K44A NOS2-/- group was significantly reduced, p<0.01, there is a statistical significance.Conclusion1. The expression of iNOS in lung squamous cell carcinoma and its surrounding microenvironment were significantly increased while iNOS can chemokine macrophage accumμlation and tumor tissue surrounding the tumor, increased tumor inflammation, thereby promoting tumor growth.2. NOS2-/- mice was observed to reduce iNOS levels can significantly reduce the incidence of tumors and prolong survival in mice.3. Bone marrow transplantation experiments demonstrated that iNOS not only can act on the organization of epithelial cells but also in macrophages can be reduced by the effect produced in the bone marrow, inhibiting lung squamous cell carcinoma.4. iNOS-/- can reduce the inflammatory response, oxidative stress and the DNA damage inhibiting the cells transformed into tumor cells, thereby reducing the incidence of cancer. |
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