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Background and Purpose malignant colon cancer is a threat to human health, the incremental rate of 4.2% per annum in the incidence of colon cancer in our country, living in a common tumor of the fourth. Combination chemotherapy is the primary means of palliative care and postoperative adjuvant treatment in patients with advanced colorectal cancer. Fluorouracil (5-fluorouracil ,5-Fu) as effective chemotherapeutic agents, either alone or jointly subsequent new chemotherapy drugs, such as irinotecan topotecan (Irinotecanpto), oxaliplatin (Oxaliplatin is), capecitabine (Capecitabine ), for the treatment of advanced colorectal cancer have achieved encouraging results, the median survival of 10-14 months. In recent years, molecular targeted drug development and clinical use of increasingly wide range of anti-vascular endothelial growth factor (VEGF) monoclonal antibodies such as bevacizumab (Bevacizumab) and anti-epidermal growth factor receptor (EGFR) cetuximab single anti-(Cetuximab, C225), the panitumumab (Panitumumab) is currently the main two types of molecular targeted drugs approved for clinical application of these targeted drugs to improve the quality of survival of patients with colon cancer. C225 is a chimeric monoclonal antibody, antagonistic EGFR binding sites point to prevent the intracellular tyrosine kinase activation in the country combined with CPT-11 has been approved as a second-line treatment of advanced colorectal cancer, in recent years, the clinical use already benefit some patients. Although cytotoxic drugs and molecular targeted drugs for some colon cancer patients to bring the Gospel, but a lot of patients with colon cancer experienced several times after chemotherapy affect the efficacy of the multidrug resistance (Multidrug Resistance, MDR), relapse after chemotherapy caused failure of chemotherapy, these are the ultimate cause of death in patients, more than 10% 5-year survival of patients with metastatic colorectal cancer is still not far. The remnants of these drugs to deal with cancer and its biological characteristics change, the impact of tumor growth and recurrence and metastasis, the key issue to be resolved by the clinical. The in vitro and in vivo studies found that the molecular mechanisms of MDR is very complex, including target gene mutations in the target gene amplification, DNA damage repair capacity differences, the drugs enter the decrease in concentration in the tumor cells. Recent studies suggest that cells remaining after chemotherapy with stem cell characteristics, tumor recurrence and metastasis is dependent on the proliferation and differentiation of these cells to achieve, at the same time a group of cells capable of expressing the angiogenic factor stimulation interstitial formation, but also cells resistance. Epithelial derived tumor cells can be provided in the role of a number of factors, lost intercellular tight junctions and adhesion links, the loss of the characteristics of epithelial cells, the morphology and characteristics of the obtained interstitial cells. Such a change is defined as the epithelial - mesenchymal transition (Epithelial - mesenchymal transition, EMT). Reduce its main characteristic changes of EMT cadherin, E-cadherin cell adhesion molecule, E-cadherin has a role in suppressing tumor invasion and metastasis. The loss of E-cadherin cells showed epithelioid features, reduce cell adhesion and thus promote tumor invasion and metastasis, tumor dedifferentiation and invasiveness. EMT is therefore one of the main factors that promote tumor cell invasion and metastasis. Colon cancer cells that remain after the drug treatment, whether EMT pathway is the focus of this project to explore local invasion and distant metastasis. The occurrence of EMT is induced by a variety of factors and the regulation of some transcription factors, growth factors, Rho family, micRNA all tumor cells can induce EMT. Has confirmed that the growth factor TGFβ family can occur through the PI3K/AKT, RAS-MAPK, Notch, Wnt signal pathway in tumor EMT, enhanced tumor invasion and metastasis. The EMT phenomenon appears subject to the TGF-β1 factor induced colon cancer cells SW-480, as a positive blank control group in vitro studies. The tumor microenvironment affect the biological characteristics of the tumor cells. Fibronectin (Fibronectin, FN) by the epithelial cells into the fibroblasts, endothelial cells, and some cancer cells secreted factors, extracellular matrix components, and its three alternatively spliced ??region are EDA, EDB and Ⅲ CS. EDA segment generally was highly expressed in many malignant tumors, but no obvious expression in benign tumors. EDA-FN, colon cancer was significantly higher than normal lacunae colon cells and their surrounding myofibroblasts, EDA-FN plays an important role in maintaining the biological characteristics of colon cancer. Serini found different sources of fibroblasts by TGF-β1 stimulation, EDA-FN and a-SM actin expression increased, presumably this way to promote migration. Colon cancer cells stimulated by TGF-β1 EMT can also be achieved through changes in the expression of EDA-FN, which is the issues need to be explored. The first part of this topic in vitro experiments, divided into three groups, the first group of applications 5-Fu treatment of colon cancer cells SW-480, the second group with the the C225 deal with colon cancer cells SW-480, the third group in combination with 5-Fu and C225 treatment colon cancer SW-480. Residual colon cancer cells were collected after treatment, drug screening residual cells (drug surviving cells, DSCs). Establish factor induced colon cancer cells SW-480, TGF-β1 appears the the EMT phenomenon of positive blank control group. Set up as a blank control group without the drug-treated cells. Contrast the detection the DSCs's EMT specific markers, and the positive blank control group. Simultaneous detection of multi-drug resistance gene, further evidence of the relationship between EMT and multidrug resistance. To investigate the EDA-FN fragment of the EMT, the third part of the in vitro experiments of the lentiviral vector construct EDA-FN gene interference, stable transfected into the normal human colon cancer cells SW-480 cells for the SW-480i, the same method empty vector transfected into normal human colon cancer cells SW-480 cells of the SW-480mock as independent of the control group, EDA-FN colon EMT specific markers to detect cells SW-480i and SW-480mock explore cancer EMT relationship. Method 1. TGF-β1-factor-induced EMT in colon cancer cell lines SW-480, as a positive blank control group. Without any treatment of human colon cancer cells SW-480 for the control group. Positive blank control group, cell shape change, compared with the blank control group were observed under an inverted microscope. Immunofluorescence staining of chemically positive blank control group in EMT-specific marker E-cadherin and Vimentin. Detected by Western blot positive blank control group cells EMT-specific marker E-cadherin and Vimentin expression. 2 were used in increments of six concentrations of 5-Fu (1,2,2.6,3.5,10,100 μmol / l) and C225 (2,6,10,13,130,260 μmol / l) continued to deal with normal human colon cancer cells SW-480 12 days, respectively, with the corresponding concentration of 6 two-drug combination treatment of human colon cancer cells SW-480. Each group used in the processing of the first four days the survival rate of the cells was determined in vitro drug sensitivity testing (MTT), and half the amount calculated inhibitory concentration (50% inhibition concentration, IC50), compared with the blank control group. Were selected close to the concentration of 5-Fu IC50 16μmol / l, and close to C225 IC50 concentration of 92μmol / l, respectively, continued to deal with DSCs morphology observed SW-480 cells after 12 days under an inverted microscope, to be compared with the positive control group and blank control group . Immunofluorescence was used to detect each set of DSCs EMT-specific marker E-cadherin and Vimentin. Collect each set of DSCs, and Western blot detection of each set of DSCs EMT specific markers E-cadherin, Vimentin and the MRP of resistance-associated protein, P-gp expression, compared with blank control group and positive blank control group. 4. Lentiviral vector construct EDA-FN gene interference, stably transfected into the normal human colon cancer cells SW-480 to obtain a cell for the SW-480i same method, the empty vector was transfected into the normal human colon cancer cells SW-480 The cells for SW-480mock, cell morphology observed under inverted microscope the SW-480mock SW-480i blank control group and positive change to do with the blank control group. The immunofluorescence staining chemical methods SW-480i, SW-480mock EMT-specific marker E-cadherin. Immunofluorescence method for the detection of E-cadherin and Vimentin expression. Western blot, cell SW-480i the SW-480mock cell E-cadherin and Vimentin expression, compared with positive blank control group. Results of the factor of 1. TGF-β1-induced human colon cancer cell line SW-480 EMT phenomenon appears inverted under the microscope to lose cell tight junctions, cell growth dispersed epithelial cells arranged start was the polarity spreading and elongation showed interstitial cell morphology. The immunofluorescence technique fluorescence quantitative analysis derived downregulation of E-cadherin, Vimentin in expression increased (P lt; 0.05), laser scanning confocal microscope, the cells showed interstitial form. On Western blot, the expression of E-cadherin down Vimentin in increased expression (P lt; 0.05), with the immunofluorescence results. 3 6 (2,6,10,13,130,260 μmol / l), increasing concentrations C225 is applied continuously processing the 12 days the survival rate of the SW-480 cells with increasing concentrations decreased, the concentration of 10,13,130,260 μmol / l, the cell viability decreased significantly, the difference compared with the untreated group (P LT; 0.05), the rate of cells found in four concentrations (81.5 ± 3.0)%, (65.4 ± 3.1)% (52.0 ± 1.8)%, (22.7 ± 0.4)%. The calculated IC50 of 85μmol / l. Continuous processing applications six increasing concentrations of 5-Fu (1,2,2.6,3.5,10,100 μmol / l) in human colon cancer cells SW-480 12 days later, cell viability with increasing concentrations of the emergence of a clear downward trend, when the concentration 2.6,3.5,10,100 μmol / l most significant difference (P lt; 0.05), compared with the untreated group. Cell viability in turn four concentrations (77.9 ± 0.2)%, (57.4 ± 1.5)%, (44.7 ± 0.3)%, (22.8 ± 1)%. IC50 was calculated 15.5μmol /. Applying the above six concentrations of 5-Fu and C225 joint processing SW-480 cells after 12 days, cell survival rates (79.4 ± 3.7) (84.1 ± 4.0) (62.3 ± 1.6)%, (44.1 ± 2.4)%, ( 22.2 ± 0.4)%, (16.9 ± 0.2)%, compared with the untreated group, the downward trend is more pronounced with (P lt; 0.05), and the results are better than with 5-Fu treatment group. Were selected nearly 5-Fu IC50 concentration 16μmol / l, and close to C225 IC50 concentration of 92μmol / l, respectively, continued to deal with the SW-480 cells in 12 days, observed significantly reduced the number of 5-Fu treated cells, DSCs morphological changes cell growth compared with dispersed epithelial cells arranged was spreading and elongation polarity showed interstitial cell morphology, C225 treated cells reduce the number of obvious, but the The DSCs same trend of transformation to mesenchymal cell morphology. And then 16 μmol / l of 5-Fu and 92μmol / l C225 combined processing SW-480 cells 12 days, found that a significant reduction in cell number material the DSCs morphology to mesenchymal transition more apparent. Chemical methods of immunofluorescence staining and fluorescence quantitative analysis showed that 5-Fu C225 group of E-cadherin decreased and Vimentin in expression increased (P lt; 0.05). Western blot assay after 5-Fu C225 DSCs E-cadherin reduce elevated Vimentin in expression (P lt; 0.05). 5-Fu C225 treated DSCs MRP and P-gp expression were higher than those of untreated normal colon cancer cells SW-480 (P lt; 0.05). Tip 5-Fu and C225 effectively kill tumor cells, and the results are better than with 5-FU treatment DSCs with EMT characteristics resistant ability. Successfully constructed EDA-FN gene RNAi interference lentiviral vector, and transfected into SW-480 cells by flow cytometry cell sorting, GFP positive rate gt; 70% of the sorting cells stably subculture, get cells SW-480i, the same method will be empty vector transfected into normal human colon cancer cells SW-480 cells of the SW-480mock as independent of the control group. Found the SW-480mock cell morphology with the blank control group did not change significantly, rules still showing epithelial cell morphology was observed under an inverted microscope. SW-480i cells with positive blank control group is similar to a typical mesenchymal cell morphology. 6 immunofluorescence staining and optical density analysis showed high the SW-480mock cells express E-cadherin and low expression of Vimentin in SW-480i cells with low expression of E-cadherin high expression of Vimentin in (P lt; 0.05). High expression of E-cadherin, Westernblot method detection of SW-480mock cells with low expression Vimentin in TGF-β1 in the SW-480i cells with low expression of E-cadherin, high expression of Vimentin (P lt; 0.05). The results showed that the SW-480i cells acquire specific marker of interstitial cells appear EMT. Conclusion 1. Colon cancer cells in 5-Fu ,5-Fu C225 under the cell viability decreased significantly, mainly 5-Fu C225. No significant decline in the survival rate of colon cancer cells in the C225 treatment alone. 2. 5-Fu C225 joint role in colon cancer cells effectively inhibit the growth of colon cancer cells significantly reduced the number of cells. The DSCs remnants of the EMT phenomenon, at the same time strong resistance ability. 5-Fu the C225 combined effects of a large number of colon cancer cells can be killed, but the DSCs have always residual resistance and the DSCs resistance and EMT. 3 EDA-FN expression after the interference of colon cancer cells, the cells appear accordingly EMT phenomenon speculated EDA-Fn may be associated with colon cancer EMT.
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