The Screening Of Different Proteins In NSCLC, The Expression Of XIAP And The Influence In The Chemotherapy

Objective:Lung cancer is one of the cancers with the highest death rate at present. One million people die caused by lung cancer each year. 80% to 85% of lung cancer is non-small cell lung cancer (NSCLC). According to the statistics, five year survival rate of lung cancer is only 10% to 15%. One of the most important reasons for the high death rate is that most of the patients only can be diagnosed at late period, which not only missed the best time of therapy, but also increased the mortality. So, it is the current focus on how to find NSCLC special tumor mark protein. A latest viewpoint was that cancer is a multifaceted disease that results from deregulated normal cellular signaling networks caused by genetic, genomic and epigenetic alterations at cell or tissue levels. Uncovering the underlying protein signaling network changes, including cell cycle gene networks in cancers, aids in understanding the molecular mechanism of carcinogenesis and identifies the characteristic signaling network signatures unique for different cancers and specific cancer subtypes. During the past several decades, the available technology to study signaling networks has significantly evolved to include such platforms as genomic microarray and proteomic analysis, which globally assesses genetic, epigenetic, and proteomic alterations in cancer. However, Pathway Array, an innovative of proteomic technology, was involved to screening the diagnostic, prognostic biomarkers and potential therapeutic targets in NSCLC.Apoptosis which also called programmed cell death or cell suicide, was a fundamental phenomena of biology in cell. In some physiology and pathology condition, it is a form of cell autonomous death controlled by genes or itself program. It is a controlled sequence of events (or program) leads to the elimination of cells without releasing harmful substances into the surrounding area. Apoptosis is indispensible to the normal growth and resistance of multicellular organism. As a main form of programmed cell death, apoptosis is an important mechanism of homeostasis in multicelluar organism. The abnormal of apoptosis is closely related to carcinogenesis, progression, and prognosis, even to the cancer drug resistance. IAPs (inhibitor of apoptosis) were a kind of conservative and specific protein of anti-apoptosis. It was a gene family and founded earliest in Baculovirus by Crook. IAPs could inhibit the infection of virus and result in the host cell death. XIAP (X- linked inhibitor of apoptosis) is the most effective protein among the inhibitor of apoptosis protein family (IAPs), which can effectively inhibit the end caspase of apoptosis, then to inhibit cell apoptosis. At present, there are a lot of researches about XIAP. It is demonstrated by many domestic and abroad researches that XIAP are with over expression among various of cancers. Its expression is obviously related to the carcinogenesis, prognosis, and sensitivity of cancer radiotherapy, chemotherapy. The paper is aiming to have a research on expression of XIAP in NSCLC and cells, its impact to the carcinogenesis, progression, and prognosis, to understand the biological characteristics.Methods and results:1. In this study, screening the different proteins in NSCLC tissue specimens were obtained from 39 patients of non-small cell lung cancer (NSCLC) who were totally removed the tumor. Pathway Array, a powerful tool, were used to analyze the protein of tissue. This is an immunoblot-based assay. The Pathway Array can assay several thousands of proteins and phosphoproteins in each sample. It has a lot of features such as sensitivity, specificity, accuracy and reproducibility. In this study, 39 proteins of non-phosphorylation were detected. The Pathway Array data was analyzed using SAM tools. 21(55%) proteins were detected. 12(32%) proteins were differentially expressed with statistical significance (p<0.05), including the over expression protein: 14-3-3-β,β-catenin,Cdc2 p34,Cdk2,Cdk4,Hsp90, Notch 4,XIAP,PCNA,and down-expression proteins: Cyclin B1,cPKCα,cdc25C.7 proteins were expression but no statistical significance, including Cdk 6, Cyclin E, p27, EGFR, Wee 1, Akt, HIF-1α. 17proteins (45%) were no expression, including Cyclin D1, BRCA1, Neu, ERK, CHK 1, MDM 2, E2F-1, c-myc, FGFR-3, Notch 1, Trap, Bcl-2, ETS1, HIF-2α, TTF-1, p53,cdc25B。And 4 proteins were differentially expressed over 4 folds (p<0.01), including Cdc2 p34, Notch 4, Hsp 90, PCNA. These differential proteins were functionally linked to cell cycle, apoptosis, cell cycle regulation, DNA repair, migration, proliferation, signaling, etc.2. Based on the above data, XIAP, a kind of protein of inhibitor of apoptosis, were selected for advanced study. The positive ratio of XIAP was 62% (24/39) in 39 cases of NSCLC, and the negative ratio 38% (15/39). The OD of density of XIAP in NSCLC tumor tissue was 0.1604±0.20334, in paratumor tissue 0.0361±0.05094.There was differentially expressed with statistical significance (p<0.01).3. We investigated the correlation of the XIAP expression with the clinicopathological parameters. It shown significant correlation between XIAP expression and tumor clinical and pathological stage (p<0.05) by X2 test analysis. There were no correlation with age, sex, smoking history and the lymphatic metastasis (p<0.05). The expression of XIAP were higher with the bad of clinical and pathological stage.4. We examine the over expression of XIAP protein in five kinds of lung cancer cell lines. Using the Pathway Array technology, the expression of XIAP was investigated in 5 kinds of cell lines. The result was over expression of XIAP in all the cell lines. And the highest cell lines of the expression of XIAP was A549.5. XIAP was related to the drug resistance in chemotherapy. In this study, to detect the expressing of XIAP mRNA in NSCLC cell line A549 by quantitative real-time PCR. The cell was treated by the different concentration of cisplatin (DDP), including 1.25, 2.5, 5, 10, 20, 40ug/ml for 24 hours, and 48 hours. The cell inhibition ratio was investigated by MTT. The ratio was higher following the increase of the concentration of DDP and the time of treatment in lower concentration (<10ug/ml). But, there were no relation in higher concentration (>10 ug/ml). The XIAP mRNA expression level was evaluated in lower concentration of DDP (<10 ug/ml). In higher concentration of DDP (>10 ug/ml), the expression was decrease.Conclusion1. Pathway Array was an innovation, powerful tool to analyze the expressed proteins with excellent sensitivity and specificity.2. There were different expression between NSCLC tumor and normal tissue. 12 proteins were differentially expressed with statistical significance (p<0.05), including the over expression protein: 14-3-3-β,β-catenin,Cdc2 p34,Cdk2,Cdk4,Hsp90,Notch 4,XIAP,PCNA, and down-expression proteins: Cyclin B1,cPKCα,cdc25C.3. Expression of XIAP was higher in NSCLC tissue. The level of XIAP expression was associated with tumor clinical and pathological characteristics.4. Expression of XIAP was over expression in five kinds of lung cancer cell lines. And the highest cell lines of the expression of XIAP was A549.5. DDP can inhibit the growth of A549 cells. There was a positive relationship between the inhibition ratio with the concentration of drug and the time of treatment. The level of XIAP mRNA expression was higher in lower concentration, and the level was decrease in higher concentration. The over expression of XIAP mRNA in lower concentration resulted by the DDP. It can inhibitor the apoptosis, and influence the chemotherapy.