A Research On Anti-tumor Activity Of Trichosanthin

Trichosanthin(TCS),which belongs to the type I ribosome inactivating protein(RIP),is the bioactive component of Chinese tradtional medicine Trichosanthes kirilowii which has been used for abortion and maintains the activity of anti-tumor.Former researches illustrate that the pathway of TCS into tumor cell is mediated by Low density lipoprotein receptor-related protein 1(LRP 1),therefore,TCS is hardly to penetrate across the cell membrane without LRP 1 and its application as an anti-tumor medicine is restrained consequently.Cell penetrating peptide(CPP)is a sort of short peptide which can translocate cargos,such as protein,nucleotide,without penetrating ability into cells.HBD is a short peptide derived from human superoxide dismutase(SOD)with penetrating ability.The recombination and expression of fusion proteins is firstly sdudied.The purified TCS maintains DNase activity together with the ability to inhibite the growth of tumor cells.Furthermore we design the recombination of TCS and HBD with the purpose of promoting the penetrating capability of TCS.Assayed by fluorescence microscope,we found that fluorescence intensity of FITC labeled TCS is lower than that of TCS with HBD,which means HBD facilitated the process of TCS crossing cell membrane.Subsequently,we construct and express recombinant vectot pET28b-TCS-HBD,and then purified fusion proteins by the virtue of His tag and Ni+.In vitro study showed that the IC50 of growth inhibitory ratio of TCS with HBD is 38?g/mL which is much higher compared with TCS which dosen't achieve an IC50 value.The result means that HBD promote the translocating ability into cells of TCS.Flow cytomertry assay,apoptotic DNA ladder electrophoresis and the observation of nuclei illustrate that recombinant protein induces apoptosis in cells.The inhibition on cancer cell A549 illustrate the increase of toxicity of TCS which gainded by HBD as well.It is the first time CPP is fused with TCS,which is indeed promoting the translocating efficiency across cell membrane.Being fused with CPP,the dosage of TCS decreased.This study has extended the application of TCS on anti-tumor.To ensure the bioactivity of fusion proteins,we also have studied the influence to fusion protein brought by different peptide linker.The quantity of EGFP fluorescence intensity and observation of penetrating ability shows that flexible linker has the minimum impact to fusion proteins.and this result supplied an important gist for the recombinant of fusion proteins.