The Study Of Immunotoxin C-CUS_245C Anti-Hepatoma Activity In Vitro

The epidermal growth factor receptor（EGFR）is strongly expressed in many tumor cells.The EGFR,closely related to proliferation and metastasis of tumor,is one of the important targets for tumor targeting therapy.Cetuximab（C）is monoclonal antibodies of EGFR,and they are clinically first-line drugs for the treatment of many malignant tumors.However,Cetuximab alone for human liver cancer cells is no obvious anti-tumor activity in vivo and in vitro,and joint use has anti-tumor activity,but not dramatically.Immunotoxins（ITs）,also named biological missile,is a set of artificial building hybrid molecules,which are capable to kill target cells,and it’s an immune complex composed of targeted part and cytotoxin（CTX）.It’s a kind of drug that can be directed against target cells.The study will make Cetuximab and Cucurmosin245C（CUS245C）by chemical cross-linking into Immunotoxins.Aim at taking advantage of targeting property of the former and the latter high anticancer activity.They can also solve some problem about Cetuximab,such as poor efficacy or side effects for liver cancer.This study will be divided into three parts: the detection of C-CUS_245C immunogenicity of liver cancer cell lines and anti-tumor activity in vivo and in vitro,so that seeking a new method for the treatment of liver cancer.Chapter 1: Cetuximab determine EGFR of human liver cancer cells.This part reports that the cetuximab determine immunogenicity of human liver cancer cells HepG2,BEL-7402,hepatic cells LO2 and breast cancer cells BT-474.By using the method of cell ELISA detecting the OD value of HepG2,BEL-7402,LO2 and BT-474,and determining their negative or positive.Using immunocytochemistry methods analyze the binding activity of cetuximab and HepG2,BEL-7402,LO2,BT–474.The results show that ELISA measured cetuximab for 1:50 0000 antibody concentration of HepG2 cells,and it is a strong positive,for 1:200000 antibodyconcentration of BEL-7402,positive,for 1:100000 antibody concentration of LO2,weakly positive,for BT-474 cells to 1:50000 antibody concentration,were negative.Measured by immunocytochemistry cetuximab effect on liver cancer cell HepG2 and BEL-7402 dyeing is clear,and the color of membrane is dark yellow.In addition,the number of positive cells is very much.The membrane of hepatocellular LO2 cell is only a few pale yellow.But the membrane of breast cancer cell BT-474 didn’t see any color.Therefore,we may draw a conclusion that cetuximab and HepG2,BEL-7402 have strong binding activity.Combined with hepatocellular LO2 are weak.But BT-474 cells have no binding activity.Chapter 2: The anti-proliferative effect of immunotoxins on hepatoma cell lines.This part reports that the anti-proliferative effect of immunotoxins on HepG2,BEL-7402,LO2 and BT-474.Using SRB analyze the anti-proliferative effect of immunotoxins on HepG2,BEL-7402,LO2 and BT-474,and using microscope records morphological changes of human liver cancer cells.In addition,SRB is used to record timeliness of immunotoxins on HepG2,BEL-7402,LO2 and BT-474.The results indicate that C-CUS_245C and CUS245 C have anti-proliferative effect on HepG2 and BEL-7402 from the microscope,but C doesn’t have obvious effect.By SRB method,we find that the IC50 value of immunotoxins have a concentration dependence and time dependence,when effected on HepG2 cell lines,BEL-7402 cell line and LO2 cell lines.And the IC50 value of CUS245 C have a concentration dependence,when CUS245 C are effected on HepG2 cell lines,BEL-7402 cell line,LO2 cell lines and BT-474 cell lines.Cetuximab alone effected on HepG2,BEL-7402,LO2 and BT-474,and inhibition rate is extremely low.Therefore,we can come to a conclusion that C-CUS_245C and CUS245 C have proliferation inhibition on HepG2 and BEL-7402,and they present the concentration dependence and time dependence.In relatively high concentrations,C-CUS_245C has anti-proliferative effect on LO2,and negative control BT-474 have not.But C doesn’t have proliferation inhibition on HepG2,BEL-7402,LO2 and BT-474.Chapter 3: Preliminary study on the mechanism of immunotoxin C-CUS_245C on HepG2 cellsThis part is to explore the mechanism of C-CUS_245C on HepG2 cells.The effect of C-CUS_245C on the protein expression of HepG2 cells was detected by Western blot,Using flow cytometry analyze the anti-proliferative effect of immunotoxins on HepG2.The results indicate that the expression of Jak2,Stat3 and Bcl-2 protein in HepG2 cells was decreased after treated with different concentration of 0 pM/L,75 pM/L,300 pM/L and 1200 pM/L C-CUS_245C for 48 h.The results of AnnexinV-FITC / PI double-staining showed that C-CUS_245C could induce the apoptosis of HepG2 cells in a dose-dependent manner.Therefore,we conclude that the immunotoxin C-CUS_245C has a dose-dependent effect on apoptosis of HepG2 cells,which may be related to Jak2-Stat3 signal transduction pathway,and by reducing Jak2,Stat3 and down-regulate the expression of Bcl-2 and Bcl-xl to induce the apoptosis of HepG2 cells.