Study On The Activity Of Novel Recombinant Trichosanthin

Trichosanthin,extracted from the root of Trichosanthes kirilowii,is a type I ribosome inactivating protein.It has two major natural activities:1)N-glycosidase activity:It can cleave the specific site of the 28S RNA,thus inhibiting the translation of protein;2)topoisomerase activity:It can change the topological conformation of DNA molecules,converting them into inactive products.Since trichosanthin has these biological activities,it has an inhibitory effect on tumor cell growth.Studies have shown that the transmembrane pathway of Trichosanthin is mediated by low-density lipoprotein-associated receptor protein 1(LRP1),so this mechanism is mainly confined to chorionic cancer,melanoma cells(LRP1+)limiting the use of Trichosanthin in anti-tumor applications.The purpose of this study is to promote the efficient transport of Trichosanthin into a variety of tumor cells to play its anti-tumor effect.The ultimate goal is to enhance its cytotoxicity against a variety of tumor cells,inhibiting tumor cell growth.Cell penetrating peptides(CPPs)have the ability to mediate the efficient transmembrane of biologically active molecules.TAT,a peptide from Human immunodeficiency virus(HIV-1),is the first discovered CPP.In this study,Trichosanthin-TAT and TAT-Trichosanthin were obtained by fusion of TAT at the C-terminus or N-terminus of Trichosanthin.The results showed that the N-terminal fusion of TAT could inactivate Trichosanthin,while C-terminal fusion of TAT had no influence.The IC50 of Trichosanthin(9.25 ?M)and Trichosanthin-TAT(6.87 ?M)indicated that Trichosanthin-TAT is more toxic than Trichosanthin.In order to obtain the recombinant proteins of Trichosanthin with higher anti-tumor ability,this study investigated the penetrating ability of human hepatocarcinoid binding domain HBP derived from HBEGF(human heparin binding-EGF-like growth factor).We attempt to fuse it into the C-terminus of the Tirchosanthin to obtain the recombinant protein Trichosanthin-HBP.The results showed that Trichosanthin-HBP had obvious cytoplasmic localization and led to significant tumor cell killing effects.The IC50 value of HeLa cells decreased from 9.25 ?M of Trichosanthin to 0.16 ?M of Trichosanthin-HBP,indicating that HBP can effectively carry Trichosanthin protein into cells.Western Blot experiments show that Trichosanthin-HBP induced more tumor cells to apoptosis by the mitochondrial pathway and death receptor pathway.In order to further improve the efficiency of drug release from the endosome,this study explored an escape strategy based on calmodulin:Calmodulin(CaM),a kind of regulatory protein,is highly expressed in humans and rodents which could promote the release of protein molecules in the endosome.It is expected that this new strategy based on CaM will help the drug to effectively carry out the esophageal escape,so that more drug molecules could enter into the cytoplasm to play the efficacy.