The Effect Of α₁-antitrypsin On The Susceptibility And Cell Biologic Behavior Of Lung Cancer

Lung cancer has been the leading cause of cancer incidence and death up to now in the world. Its mortality has been the top of all malignant tumors in citizen of China. With the development of basic and clinical medicine, great progress has been achieved in the diagnosis and treatment of lung cancer in the recent thirty years, however it is far from satisfaction in understanding the development of lung cancer, neither the outcome of therapy of the disease. Carcinoma is now considered as a polygene disease resulted from the interaction of environmental and genetic factors. In addition to focusing on prevention of environmental factors, study on genes associated with carcinoma has been the highlight in 21st century.α1AT is an important serine protease inhibitor. Previous studies have showed thatα1AT is related to chronic obstructive pulmonary disease and lung cancer in white population. The Z,S alleles are believed to be related to lung cancer susceptibility in white population, however the same result is seldom reported in Chinese Han population. So far, a controversy about the relationship betweenα1AT polymorphism and lung cancer susceptibility in Chinese Han population is still remained. Early studies have showed thatα1AT was expressed in many tumor cells and the level ofα1AT seemed to be associated with the prognosis of malignant diseases. Lacking of the findings of study, further investigation is needed to clarify the relationship betweenα1AT and prognosis of lung cancer. ObjectivesTo study the relationship betweenα1AT polymorphism and lung cancer susceptibility in Shannxi Han population; and to investigate the correlation ofα1AT expression with biologic behavior of lung cancer cells and its mechanism; additionally, to study the influence ofα1AT on A549 cell line and its underlying mechanism.Methods1. 330 blood samples were collected from Shannxi Han population including 130 cases with lung cancer, 100 cases with COPD and 100 individuals as normal control. The frequency of exonⅤ,Ⅲand 3'mutants ofα1AT was screened by PCR-RFLP to analyze the correlation of these mutants with lung cancer statistically.2. 107 specimens of lung cancer were collected including 45 with squamous cell lung cancer, 42 with adenocarcinoma and 20 with SCLC. The expression ofα1AT, CD3, Fas, FasL and DcR3 was detected by SP immunohistochemical staining to investigate the relationship betweenα1AT expression and histological type, clinical staging, cell differentiation, lymphonode metastasis of lung cancer, and to analyze the correlation ofα1AT with CD3, Fas, FasL and DcR3 statistically.3. The human lung carcinoma cell line A549 was cultured and the coding sequence ofα1AT cDNA was cloned by RT-PCR to constructα1AT eukaryotic expression vector. After the vector was constructed and the plasmids were transfected into A549 cells , and then the stable transfected cell lines were selected by G418. The cell growth curve was drawn through counting the number of cells per day and the rate of apoptosis of cells was determined by AnnexinV staining and Hoechst staining. The mRNA transcription and protein expression levels ofαlAT, Fas, FasL and DcR3 were measured by RT-PCR and western blotting. The impacts ofαlAT overexpression on the proliferation and apoptosis of A549 cells and on the expression level of Fas, FasL and DcR3 were evaluated.Results1. Although there was no significant difference between the three experimental groups statistically,Z and S alleles were reliably detected in Shannxi Han population. 3'mutant was found in 28 patients with NSCLC (21.5%) and 7 patients with COPD (7%) and the frequency of detection was significantly higher than that in control. The prevalence of 3'mutant in patients of lung cancer with COPD (42%) was significantly higher than that in patients of lung cancer without COPD (8%) with a 7.552 of odds ratio.2. The rate of positive expression ofαlAT in squamous cell, adenoid cell of lung cancer and SCLC was 80.0%, 76.2% and 40.0% respectively. There was no significant difference between squamous and adenoid cell of lung cancer, but theαlAT positive expression in SCLC was significantly lower than that in the two NSCLC groups. Interestingly, the results showed that the lower the clinical staging and cell differentiation with lymphonode metastasis was, the less theαlAT expression was compared with non-lymphonode metastasis.3. The higher level of CD3 positive expression was resulted from the higher positive expression ofαlAT in NSCLC.It was indicated that the number of tumor infiltrating lymphocytes was increased byαlAT.4. The overexpression ofαlAT was followered by an overexpression of Fas and FasL as well as a low expression of DcR3 in NSCLC. A positive correlation was betweenαlAT, Fas and FasL, but a negative was betweenαlAT and DcR3.5. A higherα1AT eukaryotic expression vector was successfully constructed and the stable transfected A549 cell line was obtained.6. The A549 cells transfected byα1AT eukaryotic expression vector growed much lower with a flat growth curve in vitro and the rate of the cell apoptosis was significantly increased than that of untransfected cells tested by AnnexinV and Hoechst staining.7. A remarkable increase ofα1AT expression of the A549 cells transfected by α1AT eukaryotic vector was detected using RT-PCR and Western blot assay. The expression of Fas and FasL was up-regulated and the expression of DcR3 was down-regulated both byα1AT in transcription and translation level.Conclusions1. Based on the results of our study, Z and S alleles were detected in Shannxi Han population, but their prevalences were not as lower as previous reports. There was still lack of enough evidence to support that the alleles were related to the development of COPD and lung cancer yet. However, the frequency of 3'mutant was higher in the patients with lung cancer or COPD. It was indicated that 3'mutant might be a risk factor of COPD and lung cancer. It was also found that the risk of lung cancer complicated with COPD was significantly increased in population carring 3'mutant. That was strongly indicated 3'mutant might be related to the susceptibility of lung cancer in Chinese Han population.2. The positive expression ofαlAT both in squamous and adenoid cell of lung cancer was obviously higher than that in SCLC. The lower was the clinical staging and cell differentiation with lymphonode metastasis, the less was theαlAT expression in NSCLC. It was indicated that the level ofαlAT expression in NSCLC was not only higher than that in SCLC, but also was closely related to the clinical staging, cell differentiation and lymphonode metastasis. Sequently we believed that the level ofαlAT expression might be a useful molecular and genetic indicator to evaluate the staging and prognosis of lung cancer.3. Being a higher expression ofαlAT in NSCLC tissues with a higher expression of CD3, it indicated thatαlAT could incease the amount of tumor infiltrating lymphocytes which is an important barrier to resist carcinoma. So we believed that the the immunity of anticarcinoma of the patients might be reflected by detection ofαlAT expression. Hopefully it would be helpful to instruct the treatment and prognosis.4. The results of immunohistochemistry study showed that the expression of Fas and FasL was up-regulated and the expression of DcR3 was down-regulated byα1AT as well. That was indicatedα1AT might play an important role in the proliferation and apoptosis of lung cancer cells.5. Theα1AT eukaryotic expression vector was successfully constructed and the A549 cells were stably transfected by this vector. The transfected A549 cells might be useful to the further study about the relationship betweenα1AT and development of lung cancer.6. The proliferation of A549 cells was obviously suppressed and otherwise apoptosis of the cells was significantly promoted due to the overexpression ofα1AT in vitro. It indicated thatα1AT might be a cancer suppressor.7. The results of immunohistochemical study were corroborated by results of molecular study in transcription and translation in which the expression of Fas and FasL was up-regulated and expression of DcR3 was down-regulated both byα1AT in vitro. Besides the role of antiprotease,α1AT could suppress tumor cell growth and promote the cell apoptosis through regulating the expression of apoptotic factors. That might be a novel explanation of the cancer suppression.