| ObjectiveColorectal cancer(CRC) is one of the most common malignancy in human beings,it is commonly and frequently diseases in our province too. CRC is the second leading cause of cancer related deaths in most western countries. Moreover, CRC occur liver metastasis frequently, the rate is 15 percent to 20 percent approximately. The valid treatment is the respectable operation, radical rate a-chieves 60 percent, but many cases still have recur or metastasis, so CRC have endangered life and health of human beings seriously. With the developing of molecular biology of oncology and others related subjects, the genes and extra -cellular biological factors related CRCsoccurance and development have become the hot spot in researchs of life science. More effective measures are taken to inhibit the infiltration and metastasis of CRC. Deep - going research for the mechanism of malignant tumor proliferation , infiltration and metastasis, some cytokines are found to take part in regulating cellular movements. The study shows that tumor cells often originate from normal cells, there are close relations between the prosesses of tumor occurrence and cellular proliferation ,differentiation or ap-optosis Through the research about oncogenesis , people found that many oncop-rotein situated different position in normal cellular signal transductional pathway, such as growth factors ,receptors of cytokines ,G - protein and so on, playing important role in promoting cellular division and proliferation. One of the most significant networks of cellular signal transduction is Ras- tyrosine kinase receptor. Almost growth factors' signal stimulated cellular proliferation , most of cyto-kine signals and lymphocyte surface combining antigen induced various reactionsdo not depart Ras - pathway. The mechanism of this pathway is that growth factor receptors have tyrosine kinase activities themselves. After ligand combines receptor , receptor' s configurative change formed dimmer so that activate intra -membranous tyrosine kinase receptor itsef phosphorylation , mean - wile substrate level phosphorylation , thus extra - cellular signal enter the cell through cell - membrance receptor and then transmit them to the efftors,, transducers and adaptors for elaborating the functions.Researches found most tyrosin kinases were oncogene protein products,c -met is one of the oncogeges, its ligand is hepatocyte growth factor ( HGF/SF, also previously termed scatter factor) , which is a unique growth factor which has been shown to exhibit mitogenic as well as motogenic properties and is a pleiotropic molecule, HGF is secreted from mesenchymal tissues, while its receptor c - met has been demonstrated on various epithelial cells. The c - met receptor protein is a tyrosine kinase receptor with an extracellular 50 - kD α- subunit and a trans-membrane 145 - kDβ - subunit.HGF/SF combine c — met can induce activation of a series of transmem-brance signal pathway and stimulate epithelioid cell' s proliferation,migration and morphogenesis. Overexpression of the c - met gene has been postulated in human gastric carcinoma,adenocarcinoma of the pancreas ,thyroidal carcinoma , cholangiocarcinoma and hepatocellular carcinoma.The differentiation and proliferation of malignant tumors are the basis of infiltration and metastasis. Many researches indicate that HGF/SF - c - met promote mitosis and induce morphogenesis, these display HGF/SF - c - met relating to proliferation ,invasion and metastasis of malignancy.Mitogen - activated protein kinases ( MAPKs) are the kinases of cell -membrance receptor and intracellular significantly regulating target objects. Cells bring extracellular signal into nucleus through this system,eliciting biological responds of cellular motivation. Cellular migration is the premise of metastasis, furthermove, the properties of tumoral infiltration and metastasis are the key factors influenced survial amd prognosis with patients.Recently,there have been reported that HGF can promote the apoptosis according to the types of cell lines. In the processes of malignant development,proliferation, differenti - ation and invasion. Regulation of cytokines and their signals transduction play critical roles. It's not clear in which transductional pathway about HGF/SF anti - apoptosis in CRC. The further research will have important significance to the proventiveness of CRC.The mechanism of HGF in the CRC is unknown uptoday. In order to explore the relationships between HGF/SF - c - met system and CRC' s proliferation ,infiltration or metastasis,we apply reverse transcription polymerase chain reaction (RT-PCR) technique, [3H] -TdR,western blotting ,cellular invasive system in vitro and MTT assay so that we can survey 1) HGF and c - met mRNA expression in CRC tissue;2) MAPKs family member ERK and p38MAPK are required for HGF - induced proliferation of CRC cell line Caco - 2 and Colo320; 3) Effect of HGF/SF and MAPK on invasion of caco - 2 and colo320 cells in Mangel invasion chamber and the expression levels of MMP - 2 mRNA, MMP -9mRNA and TIMP - 1 ,TIMP - 2 mRNA ;4) HGF and curcumin regulate apoptosis of CRC cells and the function of MAPK pathway in this process.Materials and Methods1. The expression levels of HGF/SF and c - met mRNA in colorectal cancer tissue was evaluated by RT - PCR technique.2. The expression of p42/p44MAPK , p38MAPK and c - met protein was detected by Western blot. The proliferation of caco - 2 and colo320 cells was detected by [3H] -TdR incorporation and MTT assay.3. Cell invasion assays and the expression of MMP -2,MMP -9 and TIMP - 1, TIMP - 2 mRNA were used to analyse the invasive abilities of caco - 2 cellsin vitro.4. The apoptosis of colorectal cancer cells was determined by flow cyto-mytric analysis.Results1. Expression of HGFmRNA and c - metmRNA in colorectal cancer tissuesand the relationship between the expression and the clinical pathology of colorectal cancer;the positive rates of HGF and c -met in colorectal cancer tissue were 52.5% and 80% respectively. There was no positive cases in the control group. There were statistical significances in the expression levels of HGF and c - met as compared with the control group( p <0.05 ). High expression levels of c - met related with differentiated grade and lymph node metastasis of colorectal cancer (p<0.05).2. The effect of HGF and the inhibitor of MAPK on the proliferation of colorectal cancer cells.2. 1 There were the protein expression of c - met in the detected cells by Western blot.2.2 HGF activates p42/p44MAPK and p38MAPK,20mg/ml HGF treated cells in different time points(0,10min, 30min, 1h, 3h) ,maximum activity in both is within 10min(2. 28 ±0.01 ,p <0.05).2.3 HGF'was found to significantly increase [3H] thymidine incorporation , when cells were pretreated with inhibitors of p42/p44MAPK (PD98059) , HGF — induced thymidine uptake was dimished in a dose — dependent manner ( p2.4 MTT assay found HGF elicit the growth and proliferation of Caco -2 , however, PD98059 inhibit this proliferation (72h: Control,0.76 ±0.12,HGF, 0.82 ± 0.05, PD98059,0.40 ± 0.08, p < 0.01) , but ther were not in time - dependence.3. Effect of HGF on invasion of caco -2 cells in Matrigel invasion chamber. When HGF were added to the FBS supplemented med - ium, there was a 1. 21 - fold increasing in the number of invading cells as compared with the FBS a-lone,PD98059 reduced 1.27 - fold in the number of invading cells as compared with the FBS alone. MMPs were involved in HGF - induced cell invasion: there was a conconitant reduction of expression of MMP - 2, MMP - 9 and increasing expression of TIMP - 1 ,TIMP - 2 mRNA in HGF - induced cells as shown by RT-PCR(p<0.05).4. HGF counteract apoptosis of curcumin - induced in caco -2 cells.
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