|
Background and purpose of colon cancer occurs in parts of the colon common gastrointestinal cancer, 40 years old to 50 years old age group the highest incidence of male to female ratio of 2 to 3:1. Europe and North America visceral kind of tumor incidence rate before the two, in our country just below the morbidity and mortality of gastric cancer, esophageal cancer, lung cancer and other common malignancies after radical resection of its 5-year survival rate of only 50 percent, surgery recurrence and metastasis is an important reason for his death. With the improvement of people's living standards, dietary changes, its incidence increased year by year, so early detection, early diagnosis, early treatment to prevent the development of cancer is very important. S100 protein family is a group of EF-hand calcium-calmodulin, contains at least 25 members, with a series of physiological functions, such as participation in cell proliferation, extracellular signal transduction, cell adhesion, cell power, calcium homeostasis, protein phosphorylation, activity and regulation of transcription factors such as; S100 protein family 21 members located on human chromosome lq21, the section chromosomal stability is poor, prone to chromosomal rearrangements, S100A2 gene is also located here. S100 protein family has been found that multiple members (S100A2, A4, A6, A7, A8, A9, A12, and B) abnormal expression in tumors and tumor invasion and metastasis and patient prognosis. Cyclooxygenase (cyclooxygenase, COX), also known as prostaglandin synthase peroxidase, arachidonic acid metabolism is the key rate-limiting enzyme. In a variety of cytokines, inflammatory mediators and the role of cancer-promoting agents stimulating factor, COX-2 expression was significantly up-regulated in the body of a variety of physiological and pathological processes, but also with a variety of tumor occurrence, development and metastasis. Wnt / β-catenin signaling pathway in the course of normal development and pathological pathways play an important role, is currently recognized as closely associated with the tumor is one of the signaling pathway. β-catenin signaling pathway is the key molecule in a variety of tumors were seen in the high expression of β-catenin. The project intends to study human S100A2 (hS100A2) on human colon cancer cell line HCT116 and SW480 cell proliferation, apoptosis, cycle, migration, invasion and tumor formation in nude mice, and to explore hS100A2 on cells and tumor tissue of COX -2 and β-catenin implications for the development of colon cancer hS100A2 clarify the role of providing experimental evidence. Method 1. Recombinant protein GST-hS100A2 Preparation and characterization of: calcium chloride method pGST-moluc and pGST-moluc-hS100A2 plasmids were transformed into host E. coli BL21, isopropyl-β-thiogalactoside (IPTG) induction expression, sonicated on ice bacteria, the use of glutathione - Sepharose 4B beads (Glutathione Sepharose 4B beads) Purification of GST and GST-hS100A2, by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) and Quantity One software identification and analysis of the effect of purification, Western-blot method recombinant protein, BCA method for protein quantification, 0.22μm filter membrane aliquots stored at -80 ℃ . (2) with different concentrations of GST-hS100A2 role in HCT116 and SW480 cells, MTT assay hS100A2 concentration-dependent cell proliferation. 3 Based on the above experimental select the appropriate role of the protein concentration of cells were detected by MTT cell proliferation, Hoechst33258 staining to detect apoptosis, cell cycle was detected by flow cytometry, scratch the more legitimate to detect cell migration and Transwell invasion assay changes in cell invasion. 4 amplification and identification of human S100A2 adenovirus (AdhS100A2), with appropriate infection efficiency infected HCT116 cells, collected cells were inoculated in nude mice was observed hS100A2 on HCT116 cells subcutaneously transplanted tumor growth. 5 were used RT-PCR, Western blot, immunocytochemistry assay hS100A2 HCT116 cells for COX-2 and β-catenin levels in; immunohistochemical assay hS100A2 right subcutaneous tissue tumor block COX-2 and β-catenin expression. Results 1.Hind Ⅲ and EcoRI digested plasmid pGST-moluc-hS100A2, the visible around 300bp fragment was cut with the restriction endonuclease match. SDS-PAGE analysis of the obtained recombinant protein GST-S100A2 purity of 92%, Western blot showed that the protein S100A2 antibody specificity can be identified in specific bands appeared at 36KD. BCA method 1 L broth were harvested approximately 15.7 mg protein. (2) the detected concentration range (3μg/ml to 300μg/ml), exogenous hS100A2 reduced in a dose-dependent manner in HCT116 cells and SW480 cell survival (P lt; 0.05); so selected as 100μg/ml Subsequent experiments intervention concentration; at this concentration, hS100A2 reduce time-dependent manner in HCT116 cells and SW480 cell survival (P lt; 0.05). 3.Hoechst33258 staining: hS100A2 for 48h in HCT116 and SW480 cell apoptosis rate was 3.7 times the control group and 4.2-fold (P lt; 0.05), prompted hS100A2 can promote apoptosis. 4 Flow cytometry results showed: hS100A2 treated HCT116 cells 12h G0/G1 phase when compared with the control group increased by 49.3% (P lt; 0.05), G2 / M phase cells decreased 41.6% (P lt; 0.05), prompted hS100A2 allows cells G0/G1 arrest. 5 wound healing was found hS100A2 treated HCT116 cell migration reduced: 12h and 24h scratches its healing rates were 40.7 percent lower than the control group and 19.2% (P lt; 0.05). 6.Transwell invasion was found hS100A2 reduce two kinds of cell invasion: 24h through the small chamber membrane HCT116 and SW480 cells were compared with the control group decreased 73% (P lt; 0.05) and 65% (P lt; 0.05). 7 tumorigenicity in nude mice experiments showed: hS100A2 subcutaneously transplanted in vivo can inhibit tumor growth delay its development process. AdhS100A2 infected HCT116 cells were inoculated subcutaneously into nude mice 6 days after tumor, the same rate of tumor formation, are 80%; But AdhS100A2 group of slow-growing tumor mass, 26 days were sacrificed when their tumor mass volume of the control group 10.4% (P lt ; 0.05); nude mice in each group were dissected in vivo detection of metastases. 8.RT-PCR shows exogenous hS100A2 can HCT116 cells of COX-2 mRNA decreased 78.4% (P lt; 0.05); Western blot shows exogenous hS100A2 reduces intracellular COX-2 and β-catenin protein levels wherein, hS100A2 group of COX-2 than the corresponding gray value correction control group decreased 70% (P lt; 0.05), β-catenin corrected gray value than the corresponding control group decreased 25% (P lt; 0.05); immunization cytochemistry also showed hS100A2 reduces intracellular COX-2 and β-catenin levels, consistent with the Western blot results; immunohistochemical assay showed hS100A2 reduce subcutaneous tumor tissue of COX-2 and β-catenin levels, which AdhS100A2 group of COX-2 than the corresponding average optical density of the control group decreased 78.7% (P lt; 0.05), β-catenin, the average optical density than the corresponding control group decreased 34.9% (P lt; 0.05). In vivo and in vitro experimental results. Conclusion 1. Prokaryotic induced by high purity biologically active GST-hS100A2. 2. Exogenous hS100A2 on human colon cancer cell line HCT116 and SW480 cells have inhibitory effects, including inhibition of proliferation, promote apoptosis, G0 / G1 arrest, inhibits cell migration and invasion; vivo experiments also confirmed its tumor growth in nude mice was inhibited, can delay their development process. Three exogenous hS100A2 HCT116 cells can be reduced COX-2 gene transcription, can reduce the HCT116 cells and subcutaneous tumor tissue COX-2 and β-catenin protein levels. Tip hS100A2 possibly through inhibition of COX-2 and Wnt / β-catenin signaling to exert its inhibitory effect on colon cancer.
|