| Objective:Lung cancer is a malignant carcinoma with most morbidity and mortality, more than a million new cases annually allover the world. It is very harmful to human’s health. About 80-85% of lung cancer is non-small cell lung cancer(NSCLC). Adenocarcinoma is the main pathological types of NSCLC. Lung adenocarcinoma is characterized by highly invasive,destructive growth, very easy to invade blood vessels and lymphatic walls,and with more blood and lymph node metastasis. There are many signal pathways that related with tumorigenesis,development, metastasis and recurrence. The research to tumor signal transduction pathway contributes to understand the pathogenesis of NSCLC, and provides a novel molecule target for NSCLC treatment.In view of this situation,many researches are also focus on exploring new ideas and more effective therapeutic targets for the treatment of NSCLC.Epidermal growth factor receptor(EGFR) is a tyrosine kinase active receptor.There are four structure similarly receptor molecules in EGFR family:Erb B1(EGFR), Erb B2(HER2), Erb B3(HER3) and Erb B4(HER4). EGFR is a transmembrane receptor composed with extracellular ligands binding domain, transmembrane anchoring domain and intracellular tyrosine kinase activity domain. Upon ligand binding, EGFR family proteins dimerize by receptor homo-dimerization or hetero-dimerization and subsequently activate tyrosine kinase activity. Activated EGFR family receptors then trigger a myriad of downstream signaling pathways, such as Ras/Raf/MEK/ERK,PI3K/Akt pathways, which promote cancer proliferation, invasion, metastasis and cancer angiogenesis. Studies have reported that EGFR over-expression was an adverse sign in prognosis of patients with NSCLC. EGFR overexpressin 40-80% NSCLC, and its expression is correlated with bad prognosis.Therefore, EGFR and its family members had become main candidate target molecules for targeted therapy development. At present, a lot of molecular targeting drugs against EGFR have been applied in the clinical treatment of NSCLC, but they are not very efficient and drug resistance is common. So it will be imperative to exploit more new effective targets in tumor.Caveolin-1(Cav-1) is a member of the caveolae family, it flask-shaped plasma membrane invaginations that are found in various differentiated cells are involved in many cell activities. Cav-1 is a 21~24 k Da integral membrane protein and a principal structural component of caveolae. Cav-1 is consist of178 amino acid residues, it has a unique hairpin conformation where both N-terminal and C-terminal are exposed to the cytoplasm. Residues 82 to 101 of the N-terminal(scaffolding domain)was the main functional region, this scaffolding domain can bind EGFR, and modulate the activity of EGFR tyrosine kinase. Recent studies have shown that Cav-1 is associated with development and progression of many carcinomas through regulate cell’s proliferation, differentiation, anoikis and signal transduction pathways.To investigate the role of Cav-1 in proliferation and migration of lung adenocarcinoma and further discussions on the regulation of phosphorylation,we did some researches:In this study, we constructed and cultured lung adenocarcinoma cells(GLC-82) stabling transfected with plasmid pc DNA3.1 and Cav-1. We adopted MTT, Wound healing and Transwell cell invasion assay to observe the effect of Cav-1 on the proliferation, migration and invasion in lung adenocarcinoma GLC-82 cells.Observed transformations of EGFR, p-EGFR,and ERK, p-ERK by western blot. Secondly, we observed the effect of Cav-1on proliferation of GLC-82 cells in nude mice. At last, we detected the protein expression of Cav-1, EGFR and Ki-67 in lung adenocarcinoma, analyzed the correlation. Therefore, the study is divided into three Parts listed below:Part One Caveolin-1 impacts on biological behaviour of GLC-82 cells Methods:1 Protein expression in different lung adenocarcinoma cellsCav-1 protein expression in different kinds of lung adenocarcinoma cell lines(GLC-82, H1975, A549, H1650 and H1299) were detected by western blot.2 Construction of stably transfected cell linesGLC-82 cells were stably transfected with plasmids expressing human full Cav-1 gene or empty plasmid and selected with G418, resulting in GLC-82/Cav-1 and GLC-82/pc DNA3.1 cell lines. The expression efficiency of Cav-1 was measured by real-time quantitative(RT-PCR) and Western blot.3 Proliferation assayGLC-82/Cav-1 and GLC-82/pc DNA3.1 cells were seeded in 96-well plates, respectively. After stimulating with different concentrations of EGF(0n M, 4n M, 20 n M, 100 n M) for 48 hours, MTT assay was used to measure the OD values and examine the proliferation ability of stably transfected cell lines.4 Migration assayWound healing assay was used to examine the abilities of stably transfected cell lines. GLC-82/Cav-1 and GLC-82/pc DNA3.1 cells were seeded in 24-well plates, respectively. Cells were serum-starved for 4 hours,then scratched with a 10μl pipette tip and washed with serum-free medium(SFM) to remove floating cells. Cells were divided into two groups: EGF stimulated group and no EGF group. Images of cells at the same field were taken until closure of the scratch.5 Invasion assayThe invasion assays were carried out using Transwell chamber with 10 mm diameter and 8 m m pore size polycarbonate membrane coated with matrigel. After starved with serum-free 1640 medium for 4 hours,GLC-82/Cav-1 and GLC-82/pc DNA3.1 cells were seeded into upper chamber with serum-free 1640 medium, respectively. Lower chamber were added into10% serum 1640 medium. After incubated for 24 hours, the invaded cells were fixed with 95% ice-cold ethanol and then stained with hematoxylin and eosin(HE).6 Western blottingGLC-82/Cav-1 and GLC-82/pc DNA3.1 cells were seeded in 35 mm dishes, starved with serum-free 1640 medium for 4 hours. After stimulating by EGF for different time(0min, 5min, 10 min, 30min), total proteins were extracted from cells. Western blot was used to examine the levels of Cav-1,p-EGFR, EGFR, p-ERK, and ERK.Results:1 Levels of Cav-1 expression in different lung adenocarcinoma cell lines.Western blot was used to detect Cav-1 levels in different kinds of lung adenocarcinoma cell lines GLC-82, H1975, A549, H1650 and H1975(0.12±0.00, 1.41±0.00, 0.64±0.01, 1.11±0.01, 1.67±0.00),least in GLC-82 cells.2 The expression efficiency of Cav-1 in stably transfected cell lines.The relative quantitive values of Cav-1 m RNA in GLC-82/Cav-1 cells(6.07±0.12) were significantly higher compare with GLC-82/pc DNA3.1(P<0.01). The relative levels of Cav-1 expression in GLC-82/Cav-1 cells(0.74±0.05) were significantly higher compare with GLC-82/pc DNA3.1(0.09±0.01)(P<0.01).3 Proliferation abilities of stably transfected cells.GLC-82 stably transfected cells were seeded in 96-well plates, Ovalue of each well was detected by MTT assay and statistical analyzed. Aftstimulating by 0n M, 4n M, 20 n M, 100 n M EGF, OD values of GLC-82/Cav-group(0.68±0.04,0.79±0.05,0.85±0.03,0.96±0.05) were significantly highcompare with control group(0.56±0.02,0.64±0.03,0.73±0.02,0.82±0.0according to EGF concentration(P<0.05 or P<0.01).4 Migration abilities of stably transfected cells.Migration rates of GLC-82/Cav-1 cells without EGF stimulating for 8h,16 h, 24 h, 32 h and 40h(14.01±0.78%, 20.70±0.86%, 25.23±0.75%, 31.96±0.92%, 35.07±0.31%) were significantly higher compare with GLC-82/pc DNA3.1 group(7.88±0.81%, 14.72±0.97%, 18.32±1.50%, 23.27±0.27%,26.52±1.39%)(P<0.01). Migration rates of GLC-82/Cav-1 cells(21.09±1.32%,28.67±0.94%, 33.66±0.64%, 42.42±1.43%, 50.00±0.00%) after 20 n M EGF stimulating were significantly higher compare with GLC-82/pc DNA3.1cells(10.01±0.80%, 16.08±1.01%, 21.77±1.51%, 28.42±1.49%, 34.68±2.27%).Migration rates of GLC- 82/Control cells after 20 n M EGF stimulating for more than 24 h were significantly higher compare with no EGF stimulating group(P<0.01).5 Invasion abilities in stably transfected cells.Invasion abilities in GLC-82 stably transfected cells were detected by Transwell assay, the invasion cell numbers of GLC-82/Cav-1 group(71±3)were significantly less than GLC-82/pc DNA3.1 group(38±3)(P<0.05).The expression of different proteins in GLC-82 stably cells were detected by Western blot after stimulating by EGF(20n M) for 0min, 5min, 10 min and30min, the levels of Cav-1 expression in GLC-82/Cav-1 group(0.96±0.67,1.14±0.14, 1.25±0.04, 0.83±0.09) were significantly higher compared with GLC-82/pc DNA3.1 group(0.00±0.00, 0.00±0.00, 0.00±0.00, 0.00±0.00).The levels of p Y-EGFR expression in GLC-82/Cav-1 group after stimulating by EGF(20n M) for 5min, 10 min, 30min(1.11±0.19, 1.18±0.24, 0.43±0.2)were significantly higher compare with GLC-82/pc DNA3.1 group(0.71±0.16,0.63±0.21, 0.02±0.12)(P<0.01). The levels of p-ERK expression in GLC-82/Cav-1 group after stimulating by 20 n M EGF for 5min, 10 min, 30min(0.15±0.08, 0.16±0.04, 0.93±0.09)were significantly higher compare with GLC-82/pc DNA3.1 group(0.14±0.10, 0.12±0.04, 0.13±0.03)(P<0.01).Part Two Effect of Caveolin-1 on proliferation of GLC-82 cells in nude miceMethods:All nude mice were divided into two groups: GLC-82/Cav-1and GLC-82/pc DNA3.1 group. Adjust cell concentration 1×107/ml with 1640 medium without serum. 1×106 cells were vaccinated in each one, There were 6nude mice in each group. Body weight and tumor volume were observed every three days, growth curve were drew. The mice were sacrificed and tumor tissue were harvested after 8 weeks. Tumor tissue were divided into twoproportions: one proportion use western blot to detect Cav-1, EGFR,p Y-EGFR, p-ERK and ERK protein expression; the other proportion use PV to observe Cav-1, Ki-67 and p-EGFR expression.Results:1 Model of nude miceGLC-82/Cav-1 group tumor volume and weight were both significantly larger than GLC-82/pc DNA3.1 group(P<0.01). But body weight were no significant between two groups(P>0.05).2 ImmunohistochemistryImmunohistochemistry were used to detect protein expression of the two group tumor tissue: protein expression of Cav-1 〠p-EGFR 〠Ki-67 in GLC-82/Cav-1 group were all significantly higher compared with GLC-82/pc DNA3.1 group(P<0.05).3 Western blotWestern blot were used to detect protein expression of the two group tumor tissue: Cav-1 protein expression in GLC-82/Cav-1 group(1.09±0.00,1.51±0.00, 1.40±0.00, 1.48±0.00) was significantly higher compare with GLC-82/pc DNA3.1 group(0.02±0.00, 0.09±0.00, 0.03±0.00, 0.03±0.00);p-EGFR expression(0.56±0.02,0.77±0.03,0.46±0.04, 0.59±0.01) were significantly higher compare with GLC-82/pc DNA3.1 group(0.46±0.02,0.33±0.03,0.36±0.01,0.53±0.01). p-ERK expression(1.05±0.02,1.24±0.03,1.52±0.02, 0.34±0.02) were also significantly higher compare with GLC-82/pc DNA3.1 group(0.21±0.03,0.37±0.02,0.75±0.02,0.22±0.02)(P<0.01).Part Three The expression of Caveolin-1 in lung adenocarcinoma tissue and relationship with clinicopathological featuresMethods:PV Immunohistochemistry were applied to detect the expression of Cav-1, EGFR and Ki-67 in 68 cases lung adenocarcinoma. And analysing the relationship with clinicopathological features.Results1 Expression of Cav-1, EGFR, Ki-67 in lung adenocarcinomaIn lung adenocarcinoma,Cav-1 and EGFR expressed in cell membrane and cytoplasm with brownish yellow or brown granules. The positive expression rate of Cav-1 was 51.5%(35/68), the positive rate of EGFR expression was 52.9%(36/68). Ki-67 distributed in nucleus with brownish yellow or brown granules, the positive rate of Ki-67 was 50%(34/68).2 The relationship between Cav-1/Ki-67 and clinical pathological features of lung adenocarcinomaIn 68 cases of lung adenocarcinoma tissue, there were significant relationship between expression of Cav-1 and the degree of tumor differentiation, lymph node metastasis and clinical stag, the differences were statistically significant(P<0.05), but not with age, sex and tumor size. Ki-67 was positive correlated with lymph-node metastasis and TNM stage(P<0.05),but not with age, sex, tumor size and pathology histological grade(P>0.05).3 The correlation between Cav-1 and Ki-67 in lung adenocarcinomaCoexpression of Cav-1 and Ki-67 were 23 cases in 68 while 22 cases were completely negative, they were positiveiy correlated(r=0.324, P<0.05).Conclusion:1 Cav-1 may promote lung adenocarcinoma cell(GLC-82) proliferation,migration and invasion abilities.2 In nude mice, Cav-1 significantly promoted tumor growth ability.3 Cav-1 may affect biological behaviour of lung adenocarcinoma cells through EGFR activation and regulating its down steam Ras/Raf/MRK/ERK signaling pathway.4 The expression of Cav-1 lead lung adenocarcinoma cells to poorly differentiated and promote the invasion of tumor. |
