| Part one The Expression of Caveolin-1 in breast cancer and its correlationwith clinicopathologic featuresObjective:Breast cancer is a serious threat to women’s health. Every year more than 1.2 million women are diagnosed with breast cancer worldwide. In China the incidence of breast cancer has been the most common malignant tumor of women in large and medium-sized cities. Poor prognosis of breast cancer is mainly caused by recurrence and distant metastasis.It has been well established that breast cancer is not only a biologically distinct disease, but a heterogeneous factors and signal transduction pathways regulating the complex process. Studies on tumor signal transduction pathways can make the awareness of the pathogenesis of breast cancer much better, and provide new perspective for further treatment. Along with the deepening of the research on signal transduction, the theory about signal transduction has been to reach out to all areas of life sciences. In normal circumstances, intracellular signaling molecules loacated a proper position, plays an important function of the cell. If the expression of a protein molecule increase or decrease will affect the normal physiological activity, leading to tumor. Many cancer-related protein actually may consists of a certain signal transduction pathway, with involvement of occurrence and development of tumor.Caveolae is flask-like membrane structures enriched in cholesterol and glycosphingolipids. Currently it was known that Caveolins family has three members: Cav-1(α, β), Caveolin-2(α, β, γ), Caveolin-3, and Caveolin-1(Cav-1) was the symbol protein of the structure.The expression of the Caveolins varies—some family members are expressed ubiquitously whereas others are expressed in specific tissues. Cav-1 consist of two cytoplasmic region-the variable N-Terminal and C-Terminal.The N-Terminal, mainly scaffold domain, formed by a highly conserved amino acid sequence. Caveolae not only interacts with cholesterol, but combinds with the catalytic subunits of signaling molecules,and regulates signal transduction by this area. With the development of molecular biology, we found alterations of Caveolae have been associated with numerous biological processes, including proliferation, differentiation, growth, development, survival, and drug reswastance, to name a few.. Cav-1,as its important structure protein, plays a major role by regulating a variety of molecules and signal transduction pathways in these processes.Now with increasing understanding of the role of Caveolin-1, people’s awareness has been increased, but there was still a heated controversy about tumor suppressor and tumor formation. Nevertheless subsequent studies have suggested a role for Caveolin-1 as a tumor suppressor in some tissues, including breast. However it has become a hot topic for further reveal the mechanwasm of Caveolin-1 in regulating related signaling pathways and inhibiting the proliferation of human breast cancer cells. This will provide a new target for tumor diagnosis and treatment. HER-2, also known as Erb B-2, located in chromosome 17q21, 185 KDa, was one cancer gene of the important members of the epidermal growth factor receptor family, including extracellular, lipophilic membrane-spanning region and intracellular tyrosine kinase(protein tyrosine kinase, PTK). Studies have shown that about 30% HER-2 overexpression in breast cancer associated with breast cancer incidence, development and a poor prognosis. With advances in technology and awareness of breast cancer, understanding of the occurrence and development of mechanism for breast cancer has been greatly improved. It was well known that overexpression of HER-2 play an important role in breast cancer. Monomeric HER-2, with no activity, can form a Dimer which generate the activation signal, tyrosine phosphorylation of receptor activation and activate a series of signal transduction leading to cell proliferation, differentiation, migration, invasion and resistance to apoptotic. In breast cancer studies, amplification and overexpression of HER-2 was closely related to proliferation and invasion of breast cancer, and HER-2 was highly expressed in 25%-30% breast cancer patients. In summary, the overexpression of HER-2 was closely associated with the growth and transformation of breast cancer, which can be considered as estimation of breast cancer prognosis.In cell signaling network, PI3K/Akt Pathway is a typical anti-apoptotic pathways, Akt also known as PKB, which is an important downstream target kinase of PI3 K signaling pathway. Extracellular signal-regulated kinases(extracellular SI GNA-l regulated kinase, ERK) is a significant part of extracellular signaling pathways, containing 3 typical levels of activation processes. In recent years, studies have suggested Caveolin-1 correlates with a multitude of processes in cancers, such as proliferation, apoptosis, migration, conversion and drug resistance. Combining the domestic and foreign research, we summed up the characteristics of Cav-1 and relationship between tumorigenesis development and roles in tumor. This reserch explores the Caveolin-1 involved in the occurrence, development and mechanism of breast cancer from the tissue, cellular and molecular levels. Further discussions on the regulation of phosphorylation of HER-2 Caveolin-1 and roles in the Ras/Raf/MEK/ERK and PI3K/Akt signal transduction pathway bases on Immunohistochemistry, plasmid transfection, Western blot, ect. Further studies are needed to validate functions of Caveolin-1 in breast cancer thus provides an effective strategy for the treatment of mammary carcinoma.Methods:To explore biological behavior of Caveolin-l and its significance in prognosis of breast cancer, 50 cases, including different levels of differentiation invasive breast cancer and normal breast tissue, have been detected by Non-biotin enzyme II(PV) Immunohistochemistry method. Adopting normal breast tissue or breast benign hyperplasia as negative control was in order to understand the expression of Caveolin-l and phosphorylation of human epidermal growth factor receptor 2(p-HER-2).1 Non-biotin enzyme II(PV) immunohistochemical method was used to detect levels of Caveolin-1 expression in breast cancer cells.Immunohistochemical results show positive Caveolin-1 mainly in the cytoplasm of epithelial cells of the breast, yellow-brown granules. According to the criteria, the expression of Caveolin-1 in normal breast tissue(80%) was stronger than in invasive breast cancer cells(32%), comparing the two statistically significant differences P<0.05.2 Relationship between Caveolin-l expression and clinicopathological factors in breast cancer.The result indicates positive Ki-67 mainly in the nucleus of mammary epithelial cells and HER-2 positive expression predominantly nucleus expression, showing a yellow-brown granules. Levels of Caveolin-l expression depressed along with lymph node metastasis appeared and lower degree of differentiation in invasive breast cancer(P<0.05). There was no association between levels of Caveolin-l expression and age, tumor diameter, ER/PR, HER-2, Ki-67 and menstruation in invasive breast cancer.3 Caveolin-l and p-HER-2 expression in breast cancerP-HER-2 predominantly expressed in membrane of the epithelial cells of the breast, yellow-brown granules.The expression of P-HER-2 was higher in Caveolin-l less expression groups and lower in Caveolin-l stronger expression of groups, with statistically significant differences(P<0.05).4 Correlation of expression of Caveolin-1 and prognosis of breast cancerThe result reveals expression of Caveolin-1-positive patients with 5-year survival rate was significantly higher than the Caveoiin-1 negative groups. Results showed that the prognosis of Caveolin-1-positive cases was significantly better than Caveolin-1 expression negative cases. Part two Influence of Caveolin-1 on biological behaviour in breast cancerMethods:1 Cells and cell cultureHuman breast cancer cell lines MDA-MB-453 were cultured in RPMIResults: 1640 culture medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 IU/ml streptomycin, at 37°C in a humidified atmosphere of 5% CO2.2 PlasmidsWe constructed plasmid pc DNA3.1-Cav1 stable transfected cells and pc DNA3.1 negative control respectively by QIAGEN plasmid extraction kits. Setting up MDA-MB-453, pc DNA3.1-Cav1(Cav-1) and transfection pc DNA3.1 transfection control groups, we obtained MDA-MB-453/Cav-1 and MDA-MB-453/pc DNA3.1 after untransfected cells induced deaths through puromycin resistance.3 Western blotting and Real-time PCRWhen MDA-MB-453、MDA-MB-453/pc DNA3.1 and MDA-MB-453/Cav-1 cells covered 80% of culture dish,we continued to culture the cells with RPMI-1640 culture medium without serum for 4 hours. Western blotting was carried out to detect levels of Caveolin-1 expression among three groups by collecting total protein.Total RNAs were isolated from stable transfed cells, MDA-MB-453/Cav-1, MDA-MB-453/pc DNA3.1 and MDA-MB-453 cultured in 35 mm dishes. The expression levels of Caveolin-1 m RNA were examined by Real-time PCR at last.4 The proliferation abilities of stable transfected cells were detected by MTT assay.MDA-MB-453/pc DNA3.1, MDA-MB-453/Cav-1 were cultured and divided into four groups stimulated for 0n M, 4n M, 20 n M and 100 n M by EGF after 24 hours. MTT assay was used to examine the proliferation, abilities in stably transfected cells of each group.5 Scratch assayMDA-MB-453/pc DNA3.1 or MDA-MB-453/Cav-1 cells were seeded into 24-well plates. Each kind of cells was divided two groups: EGF-free groups and 20 n M EGF-stimulation groups, respectively.After scratch a line on the dish using a sterile 200 ul pipet, the cells have been observed at 0h, 24 h, 48 h, 72 h and 96 h hours later and calculated Cell migration rate separately.6 Matrigel invasion and transmigration assaysFor Matrigel invasion assay, MDA-MB-453/pc DAN3.1 and MDA-MB-453/Cav-1 were stained HE and seeded into Matrigel-coated Transwell insert supplemented with RPMI-1640 culture medium without serum for 4 hours. Plates were imaged under microscope and counted the penetrating cells.Results: 1 The expression of Caveolin-1 in different cells. 1.1 The expression of Caveolin-1 was assessed by Western blot.Statisticed the Caveolin-1 expressions of MDA-MB-453 、 MDAMB-453/pc DNA3.1 and MDA-MB-453/Cav-1 as follows: 0.02±0.00,0.02± 0.00,0.60±0.03. After stably transfected, the expressions of Caveolin-1 in MDA-MB-453/Cav-1 group(0.73±0.01, 0.373±0.05) were significantly higher than control group. There was statistical difference between two group(P<0.01). 1.2 Proliferation abilities in different cells.The OD value of each well was measured by MTT colorimetric assay, after EGF stimulating for 0n M, 4n M, 20 n M and 100 n M, respectively. OD values of MDA-MB-453/Cav-1( 0.80 ± 0.07, 0.86 ± 0.03, 0.93 ± 0.03, 1.00 ± 0.03) were significantly higher than the control groups(0.93 ± 0.05, 1.02 ± 0.04, 1.12 ± 0.06, 1.32 ± 0.09). There was statistical difference between two group(P<0.01). 2 Migration abilities in MDA-MB-453/Cav-1 cells.Measuring scratch width using appropriate software, cell migration rate is calculated.(Cell migration rate =(initial width- current width)/2/scratches x100% initial width). Migration rates of MDA-MB-453/Cav-1 cells without EGF stimulating for 24 h,48h,72 h and 96h(2.31±0.55,9.04±2.14,16.08±2.54,21.93±2.60) were significantly lower compare with control group(5.21±0.42, 12.13±0.38, 23.56±1.99, 35.04±2.47)(P<0.05). Migration rates of MDA-MB-453/Cav-1 cells after 20 n M EGF stimulating were significantly higher compare with no EGF stimulating group. Migration rates of MDA-MB- 453/Cav-1 cells after 20 n M EGF stimulating( 4.80±0.48, 11.90±0.98, 19.50±2.53, 27.71±2.06)were significantly higher compare with no EGF stimulating group(2.31±0.55, 9.04±2.14, 16.08±2.54, 21.93±2.60)(P<0.01). 3 Invasion abilities in two different stably transfected cells.Transwell assay for detection of cellular invasion, the MDA-MB-453/Cav-1 group(16 ± 2), significantly lower than the control group(31 ± 3), considered statistically significant.(P<0.01) Part Three Effect of Caveolin-1 on activation of HER-2 in breast cancer cellsMethods: Western blot was assessed the expression of Caveolin-1, 4G10, HER-2, p-Akt, Akt, p-ERK, ERK and β-actin stable in stably transfected breast cells.Samples were divided into two groups: pc DNA3.1-Cav1 stably transfected group and pc DNA3.1 control group. Screened stably transfected MDA-MB-453/pc DNA3.1 and MDA-MB-453/Cav-1 breast cancer cells by Puromycin, after EGF stimulating for 0, 5, 10, 30,min, extracted proteins respectively. Determined the expression of Caveolin-1, 4G10, HER-2, p-Akt, Akt, p-ERK, ERK and β-actin stable in stably transfected breast cells by Western blot.Results: Effect of overexpression of Caveolin-1 on activation of HER-2.The results of statistics demonstrated that levels of Caveolin-1 expression the pc DNA3.1-Cav1 groups(1.12 ± 0.03, 1.08 ± 0.06, 1.13 ± 0.06, 1.11 ± 0.04) is significantly higher than the control group(0.05 ± 0.01, 0.04 ± 0.01, 0.04 ± 0.00, 0.04±0.01)at every tested point; Expression levels of HER-2 phosphory lated by pc DNA3.1-Cav1 group after EGF stimulated respectively(0.98± 0.04, 0.50 ±0.03,0.20±0.01) were significantly lower than control group(1.25± 0.03, 1.07±0.03, 0.50±0.02); The level of Akt in phosphorylated pc DNA3.1-Cav1 group(0.50 ±0.01, 0.67 ± 0.03, 0.42 ±0.01)were significantly lower than control group(0.85±0.05, 1.20±0.08, 0.82±0.04) after EGF stimulated; Expression levels of ERK in phosphorylated pc DNA3.1-Cav1 group(0.58±0.01, 0.80±0.03, 0.45±0.01) were significantly lower than control group(0.93±0.02, 1.10 ± 0.06, 0.72 ±0.05).There was a statistically significant(P<0.01).Conclusion:1 Caveolin-1 expression is greatest in normal breast epithelial cells, lower in invasive breast cancer tissue,furthermore, correlates with malignant increment. In invasive breast cancer, the expression of Caveolin-1 is negatively correlated with the expression of p-HER-2.2 Caveolin-1 expression levels inhibit breast cancer cell proliferation, migration and invasion, suggesting that Caveolin-1 on breast cancer cell proliferation and metastasis inhibition.3 Overexpression of Caveolin-1has a great influence on signal transduction pathway in breast cancer cells Increased expression of Caveolin-1 can reduce the level of phosphorylation of ERK and AKT. Cav-1 may promot the growth of breast cancer cells via enhancing EGF-induced HER-2 activation(phosphorylation) and downstream signal transduction. |
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