Induces The Human Colon Carcinoma Cell Line Lovo Be Resistant To Raltitrexed And The Investigation Of Some Resistant Related Proteins

Background and purpose:The carcinoma of large intestine is one of the most common malignant tumor of the digestive system.Chemotherapy is the important factor in the combined therapy of this neoplasma.The antifolates such as 5-Fu is the basic agent in the chemotherapy program to colorectal carcinoma. ZD1694(raltitrexed)is a novel antifolate antimetabolite which was developed by the Zeneca corporation for the treatment of the advanced stage of colorectal cancer.From the dates of clinical application and laboratory research,it is a potential agent because of its good curative effect and tolerated adverse effect. So to investigate the resistance mechanism of tumor cells to raltitrexed has sense not only to development of new drups but also to make itself be used better.The object of this study is to induce the human colon carcinoma cell line lovo to be a resistant one to raltitrexed,then use immunohistochemistry and western blot to study whether Pgp,MRP,LRP and GST-Ï€are involved in the resistance process or not.Methods : The lovo cells were established by repeated,intermittent exposure to escalating concentrations of raltitrexed.The first time,lovo cells was treated with 200ug/ml raltitrexed for six hours,more than 95% cells were dead then allowed to grow in drug-free medium.After thirty five days ,the survival cells grew to nomal state which were then treated with 400ug/ml raltitrexed for another six hours,after grew 28 days in the drug-free medium,the lovo cells recovered to common condition.The third time,the raltitrexed concentration is still 400ug/ml,treated for six hours and it took 25 days for the surviving cells togrow to confluence.After subculture,a flask of cells were selected once more with 400ug/ml raltitrexed for six hours.The above established cells were called lovo/raltitrexed cells.We observed the shape of this two cell lines,and compared their doubling generation times and growth curves.Lovo and lovo/raltitrexed cells were evaluated with the MTT assay which measures mitochondrial activity and to determine the concentration reguired for 50% inhibition of growth.Use immunohistochemistry to observe whether Pgp , MRP , LRP and GST-Ï€expressed both in this two cell lines or not.Besides,we use western blot to check the variance of this four proteins between them in order to find which one plays a role in the resistance process.Results:1. Use high dose pulse treatment selection to obtain the ZD1694 resistant cell line named lovo/raltitrexed cells which were treated for four times in about four months.The final level of resistace to raltitrexed of lovo/raltitrexed was 10-fold compared to parental lovo cells.2. Love and lovo/raltitrexed cells have almost same shape and both be fusiform.Compared to parental love cells, lovo/raltitrexed cells have a same growth curve,indicated that their growth rate were fast as same as lovo cells,although it looked just a little slower than parental cells.3. Use Immunohistochemistry to analyze this two cell lines indicates that both of them express Pgp,MRP,LRP and GST-Ï€.4. Western blot analysis shows lovo/raltitrexed cells express Pgp,MRP and LRP more than lovo cells,expecially Pgp and MRP.Expression of LRP in this two cell lines are the same.Conclusions:1. The lovo cells could be induced in vitro to be the acquired raltitrexed-resistant lines.2. High dose pulse treatment selection is a effective way to establish the resistant cell model.3. The lovo cells are sensitive to raltitrexed in vitro.The excretion mediated by membrane proteins Pgp and MRP plays a key role in the resistance process.4. The metabolic enzyme GST-Ï€also takes part in this procedure.LRP has nothing to do with this process.