A Study On The Role Of α₁-antitrypsin In The Development Of Lung Cancer

Carcinoma of the lung has been the leading cause of cancer incidence and death up to now. 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, however it is far from satisfied in understanding the development of lung cancer, neither the outcome of therapy of the disease.α₁ AT, an important serum protease inhibitor, has been found to be synthesized by many malignant carcinoma cells. It is reported that α₁AT locally suppressed tumor cell proliferation, metastasis and angiogenesis in the solid tumors. So it is believed that the α₁AT might play an important role in the diagnosis and treatment of the tumors.Objective: To investigate the expression of α₁AT in non-small cell lung carcinoma (NSCLC) and its effect on cell proliferation in human lung adenocarcinoma cell line A549.Methods:1: α₁AT expression was detected using Envision immunohistochemical staining in 72 specimens of the NSCLC prepared with paraffin embedding.2: The human lung cancer cell line A549 was cultured and the coding sequence of α₁ AT cDNA was cloned by RT-PCR to construct α₁AT eukaryotic expression vector. When the vector was constructed and selected by G418, A549 cells were transfected with pcDNA3.1(+)(as control)vector or pcDNA3.1(+)-α₁AT. These cells were cultured on cover slips and observed respectively through HE staining and electron microscopy. All the cells were cultured and counted every other day, thus the growth curve was drawn. α₁AT mRNA and protein levels were measured with RT-PCR, immunofluorescence microscopy and western blotting. Apoptosis was detected by AnnexinV staining and Hoechst staining.3. BALB/c nude mice were injected subcutaneously with A549, A549-pcDNA(3.1+), A549-pcDNA(3.1+)-α₁AT cell suspension respectively to construct nude mouse lung carcinoma animal model. The formation and proliferation of the tumors were observed by measuring the tumor volume and weight.Results:Part I : α₁AT protein expression was observed in 66 of 72 cases of NSCLC. It was highly expressed in lung cancer tissues (91.6%) than in para-carcinoma tissues (41.6%)(P<0.05), which may be due to the synthesis of NSCLC cells themselves. aiAT level was much more higher in stage I—II cases than in stage III-IV cases, which might imply that α₁AT was related with lung cancer clinical staging. Positive correlation was found between the expression and the cell differentiation. The lower was the tumor cell grade, the less was α₁AT expression; There was no significant difference betweenmoderate- and high-differentiated NSCLC samples in the expression, but it was found that staining was more intensive in later group. Negative correlation was found between the expression and the tumor size. The bigger was the tumor the less was the expression. There were no correlation between α₁AT expression and sex, age and NSCLC histologic types.Part II: cDNA coding otiAT about 1200bp was generated by reverse transcription-PCR. The purified PCR product was inserted into the BamH I and EcoR V sites of pcDNA3.1(+). Sequence analysis showed that it was the same as the α₁AT sequence reported. Then α₁AT PCR product was cloned reversal into pcDNA3.1(+) vector. The A549 stable transfectants with either control vector pcDNA3.1(+), or pcDNA3.1(+)-α₁AT were established. Morphology of A549/ pcDNA3.1(+)-ctiAT under electron microscopy displayed signs of apoptosis, such as plasma membrane blebbing and multiplied nuclei. Cell growth curve showed A549/pcDNA3.1(+)-α₁AT proliferated slowly than other kinds of cells.0.5 × 10⁵ different kinds of cells were cultured in 6-well plates respectively. In day 7, the number of A549/ pcDNA3.1(+)-α₁AT was just 3.12 × 105 but all other cells was 6.83 × 105. A549/pcDNA3.1(+)-aiAT had significantly decreased in cell number compared with the other two groups.Strong and diffuse green staining of α₁AT emerged in pcDNA3.1 (+)-α₁AT transfected cells by indirect immunofluorescence staining, but in negative control and the native A549 the staining was very weak or absent.The mRNA of α₁AT in A549 and A549/ pcDNA3.1(+) cells were nearly undetectable by RT-PCR. In contrast, the mRNA level of α₁AT was detected obviously in A549/pcDNA3.1(+)-α₁AT cells. At the same time , β -actin mRNA had not been changed in all kinds of cells. The similar change wasfound in the level of α₁AT protein through western blotting.The pcDNA3.1(+)-α₁AT transfected cells showed more typical apoptotic characters, such as shrinkage nuclei, chromatin condensation, plasma membrane blebbing and chromatin breakage fragments by Hoechst staining under fluorescence microscope than the negative control and the native A549 cells. The same scene was found under transmission electron microscope. The apoptosis rate in the pcDNA3.1(+)-α₁AT cells was significantly higher than that of the negative control and the native A549 cells. A549 cell's apoptosis was increased significantly after transfected with α₁AT.Partlll: When tumor cells were seeded in nude mice, the tumorigenesis was found in day 16 in those injected with A549/ pcDNA3.1(+)-α₁AT cell suspension transfected by α₁AT gene , as the other two groups was in day 7. The incubation period of tumors was lengthened in the experimental group than in the control and the tumor volume was significant decreased either. The tumor-inhibiting rate was 55.5%. It indicated that α₁AT could inhibit thesubcutaneous tumorigenic of A549 in nude mice. Conclusion1.Our study showed that the expression of α₁AT in NSCLC tissues were significant higher than in para-carcinoma tissues, and the expression of α₁AT was not correlated with the age, gender and histological types. Its expression level was negatively correlated with NSCLC clinical TNM staging but positively with cell differentiation. The lower was the tumor cell differentiation and the clinical TNM staging, the less was α₁AT expression. On the other hand, the smaller was the tumor size, the higher was the expression, which could also be found in clinical staging. So α₁AT might be an index for estimating the degree of malignant and progression of the...