Preliminary Study On Antitumor Effect Of Recombinant Trichosanthin And Its Modified Derivatives

ObjectiveRecombinant trichosanthin(rTCS)and its mutant with the reactive sulfide group(rTCS-N10-C)was prepared by genetic engineering technology.The rTCS-N10-C was chemically conjugated with LMWP,PEG,or BSA to produce the rTCS-N10-SS-LMWP,rTCS-N10-PEG,and rTCS-N10-BSA,respectively.The in vitro antitumor activity of rTCS and its modified derivatives was investigated.MethodsThe plasmid of rTCS was constructed at first,and then its mutant was prepared by introducing a strategically designed amino acid sequence(the substrate sequence of legumain plus cysteine,NNNNNNNNNNC)to the C-terminal through recombinant technology to express rTCS-N10-C.The plasmid of rTCS or rTCS-N10-C was transfected into E.coli BL21(DE3)for expression.The protein products were purified by using a Ni2+-NTA column and heparin column.The rTCS-Nio-SS-LMWP,rTCS-N10-PEG and rTCS-Nio-BSA were prepared through the C-terminal active sulfide group of rTCS-N10-C with the activated LMWP,PEG,and BSA,respectively.The thus-formed conjugates were purified with fast protein liquid chromatography(FPLC),characterized by SDS-PAGE.The protein content was determined by BCA method and the antitumor activity measured by MTT and flow cytometry apoptosis assay.ResultsThe rTCS and rTCS-N10-C were successfully prepared through genetic engineering technology and the rTCS-N10-SS-LMWP,rTCS-N10-PEG,and rTCS-Nio-BSA were synthesized by chemical coupling method.The in vitro studies showed that rTCS could inhibit tumor cell proliferation and induce significant early apoptosis in both sensitive tumor cells and drug-resistant tumor cells.The antitumor activity of rTCS-N10-SS-LMWP was significantly higher than that of rTCS.Legumain could hydrolysis the peptide bond of asparagine of rTCS-N10-PEG,and then the rTCS would be cleaved from PEG.The rTCS-N10-PEG could induce significant early apoptosis in both tumor cells and drug-resistant tumor cells,although it had not obvious cytotoxicity on tumor cells by MTT assay.The rTCS-N10-BSA caused a concentration-dependent inhibition of tumor cell growth.Animal experiments revealed that rTCS have high antitumor activity,while the antitumor activity of rTCS-N10-BSA was silghtly lower than rTCS in vivo.ConclusionThe rTCS exhibited strong antitumor activity.Modification with LMWP can significantly improve the antitumor effect of rTCS.It is a feasible way to research traditional Chinese medicine by modern techniques.