The Inhibitory Effect Of Trichosanthin On Growth Of Colon Cancer Cell And Its Molecular Mechanism

Colorectal Cancer is one of the most common malignant tumors of digestive system in human being,and the incidence rate is ranked third among human malignant tumors.Colon cancer is prone to metastasize to distant liver tissue leading to the death.At present,although surgical resection is the most common treatment for colorectal cancer,chemotherapy is also an important tool for controlling the disease and improving the quality of life.The occurrence of malignant tumor is related not only to the abnormal proliferation of cells,but also to the apoptosis of cells.Therefore,apoptosis plays an important role in the control of tumor growth.Pharmacological studies reveal that some traditional Chinese medicines have a potent effect on inhibiting tumor growth.Trichosanthin(TCS),a traditional Chinese medicine,is extracted from the root of herb trichosanthes.It is a type I ribosome inactivating protein with RNA N-glycosidase activity specifically hydrolyzing the eukaryotic 28S ribosomal rRNA specific adenosine.Studies show that it posses anti-tumor,anti-virus and other pharmacological activity.However,its molecular mechanism of anti-tumor activity is still unclear.In this study,we explored whether TCS could inhibit colon cancer cell growth and how TCS exerts its biological function in colon cancer cell.Our results showed in following four aspects.(1)TCS inhibited the growth of HCT116 colon cancer cell.HCT116 cell showed increased shrinkage and deformation in morphology with obvious drug concentration and time dependence.(2)Treatment with TCS blocked HCT116 colon cancer cell in S phase and induced apoptosis of HCT116 colon cancer cell.(3)Western blotting analysis showed that expression of procaspase-3/procaspase-9/PARP was decreased.The expression of pAkt was increased,while the change of total Akt protein was not obvious.(4)The wild-type TCS inhibited the luciferase activity,while the mutant Y70A did not inhibit the luciferase activity in the cell.Interestingly,the mutant R163Q showed stronger inhibitory effect on luciferase activity than that of the wild-type TCS.Part ⅠThe inhibitory effect of TCS on the growth of colon cancer cell and its molecular mechanismObjective:To investigate effect of TCS on the growth of colon cancer cell and its molecular mechanism.Methods:The effects of TCS treatment were examined in HCT116 colon cancer cell.(1)The Invert microscope was used to observe the effect of TCS on the morphology and proliferation of HCT116 cell.(2)The flow cytometry was employed to detect cell cycle and apoptosis.(3)Western blotting was performed to check the expression of caspase-3,caspase-9,PARP,pAkt and Akt.Results:The results showed that TCS inhibited growth and proliferation of HCT116 colon cancer cell.(1)TCS inhibited the growth of HCT116 colon cancer cell.HCT116 cell showed increased shrinkage and deformation in morphology with obvious drug concentration and time dependence.(2)Treatment with TCS blocked HCT116 colon cancer cell in S phase and induced apoptosis of HCT116 colon cancer cell.(3)Western blotting analysis showed that the expression of procaspase-3/procaspase-9/PARP was decreased.The expression of pAkt was increased,while the change of total Akt protein was not obvious with the treatment of TCS.Conclusions:TCS could inhibit the growth of colon cancer cell,which is positively correlated with drug concentration and time.TCS could block colon cancer cell in S phase and induce apoptosis of colon cancer cell.The activation of Caspase-3 and caspase-9 may mediate the pathway of TCS induced apoptosis in colon cancer cell.The results suggest that it may be related to the up-regulation of PI3K/Akt pathway.Part ⅡBiological activity of the wild-type trichosanthin and its mutants Y70A and R163QObjective:To investigate the biological activity of the wild-type TCS and its mutants Y70A,R163QMethods:Mutants Y70A and R163Q TCS were constructed by site directed mutagenesis.293T cell was transfected with pGL-3 alone,or in combination with the wild-type TCS,its mutants Y70A and R163Q.Luciferase activity was compared between different groups.Results:The wild-type TCS inhibited the luciferase activity,while the mutant Y70A did not inhibit the luciferase activity in the cell.Interestingly,the mutant R163Q showed stronger inhibitory effect on luciferase activity than that of the wild type TCS.Conclusions:The wild-type TCS and mutant R163Q are active in inhibiting ribosomal protein synthesis in the cell.The inhibition capacity of mutant R163Q is stronger than that of wild-type TCS.The mutant Y70A do not have the inhibition capacity of ribosomal protein synthesis in the cell.The results suggest that Y70 and R163 are important amino acids for the enzyme activity of TCS.