Significance And Mechanism Of FasL And Assosiation Proteins In Hepatic Metastasis Of Colorectal Carcinoma

Colorectal Carcinoma(CRC)is one of the commonest carcinoma in our country.Its morbidity and mortality rates are the second top diseases in carcinoma of the digestive tract. Though diagnosis and treatment techniques is increased,the morbidity of CRC is increasingly higher year by year in recent years, which is due to local recurrence and live metastasis. The key point of improving the 5-year morbidity of CRC is how to find, diagnose and treat earlier, and to prevent postoperated CRCs from recurring and metastasizing. So it is very important to investigate the mechanisms of its recurrence and metastasis. Our purpuse is to search some new, specific and hypersensitive makers to diagnose its recurrence and metastasis.Aims To adapt the yeast two-hybrid system for the study of proteomics in the field of the fetal liver library, and to screen and verify the proteins which interact with FasL. By animal model which tend to liver metastasise,to verify its expression rules in their cells and tissues. To study the mechansim of FasL and its interacting proteins in CRC local recurrence and liver metastasis,and to provide theoretical evidence for screening some earlier diagnosing makers and some molecular treating targets in the liver metastasis of CRC.Methods Amplify FasL-pGBKT7 vector, then use Western'Blot to verify FasL protein express in yeast AH109 cells. By transferring FasL-pGBKT7 and liver cDNA library genes into yeast AH109 cells,then by high stringency screen, we got and verify FasL interacting proteins. By animal model which tend to liver metastasise, compare COLR-1 cells with HR-8348 cells in the tumor cells character.By immunohistochemistry, examine different nude mice(liver metastasis or none liver metastasis) tissues to analyze the expression diffences of these proteins,which is to verify the mechanism of liver metastasis. All data is anlysized by SPSS 12.0 software. Results FasL-pGBKT 7 has no auto activity in AH109 cells,and can express FasL protein.We screened 4 proteins that interact with FasL protein, including metallothionein 1K,1G, cathepsin B and anchor attachment protein. In the model of CRC subcutaneous tumor transplantated to liver,then cultured in vitro, we screened COLR-1 cells which have the potential of metastasizing to liver. COLR-1 cells have the more tumor character compare with HR-8348 cells. Flow cytometry analysis showed that the PI rate of COLR-1 cells is much more higher than HR-8348 cells. By the means of immunohistochemistry, we have examined the expressions of FasL and cathepsin B in COLR-1 cells,it shows that cathepsin B expresses much higher in liver metastasis tissues(82.35%)than that in none liver metastasis tissues, and FasL expresses high in both the tissues.Western blotting shows that both FasL and cathepsin B enhancedly express in COLR-1 cells.Conclusions We identified the reliablility and repeatability of the interaction between FasL interaction proteins by yeast two-hybrid. The four interaction proteins play the important roles in the study of recurrence and matastasis of cancer research, particularly are cathepsin B and anchor attachment protein. Cathepsin B can give the tumor cells the potention to metastasis and invading. Anchor attachment protein is blank in the field of CRC research. Cathepsin B is verifid to interact with FasL, and increasingly express in vivo of the liver metastasis animal, which means that FasL infects its backward position proteins,include Cathepsin B, active them to increasingly express in CRC cells. Then Cathepsin B act on the proteins in the basement membrane of liver cells, promote the CRC cells metastasize to liver, enhance the capability of invading and metastasis.The results give the important rules of the recurrence and liver metastasis of CRC cells.