Isolation, Puirfication And Characteirzation Of Anti-tumor Protein From Wheat Germ And Its Anti-tumor Mechanism

As a byproduct of wheat flour processing, wheat germ is rich in high quality protein,which tends to have good biological activity. This provides an important direction for the deepdevelopment and efficient use of wheat germ. Cancer has been one of the serious diseaseswhich endanger human life and health today, and looking for anti-tumor activity componentsfrom plants has become a hot issue. Since the anti-tumor activity of wheat germ has beenproved, it is very meaningful to find the anti-tumor ingredient and investigate the mechanism,which may provide some ideas on exploiting anti-tumor functional foods or drugs from wheatgerm.Firstly, a novel anti-tumor protein (WGWSP11) was separated from water-soluble extractof defatted wheat germ by fractional precipitation with ammonium sulfate, ion-exchangechromatography on DEAE-Sepharose Fast Flow and gel filtration chromatography onSuperdex200. The cell culture experiments in vitro indicated that WGWSP11had effects onthe cell viability of breast cancer cell line MDA-MB231, human gastric cancer cell line SGC-7901and human colon cancer cell line HT-29,.Especially the strongest effect on breast cancercell line MDA-MB231was observed. The purity was identified by high performance liquidchromatography（HPLC）and the result showed that it was a single symmetrical peak. SDS-PAGE showed that it was a single band, its molecular weight was approximately41kDa andit is a glycoprotein by PAS staining.Secondly，the results of near-UV CD was in accordance with the results of thefluorescence emission spectra of WGWSP11in ethanol solution and GuHCl solution, whichshowed that there was only Trp residues on the surface of WGWSP11molecular. Far-UV CDshowed that the α-helix, β-sheet, β-turn and random coil content were20.69%,13.18%,50.24%and15.99%, respectively. The protein was a kind of glycoprotein revealed by FTIRand it was O-glycosidic bond revealed by β-elimination reaction analysis. The N-terminalamino acid sequence detected by LTQ was：MDKFKGAVMVGALRLFALLPWRAVQWTGTAIGWLMWKLPNRSREVARINLSKCFPELSEPELEKLVGRSLMDIGKTLTESACAWIWPAQKSIDLVREVEGLDVLKDALASGKGVVGITSHLGNWEVLNHFYCSQCKPIIFYRPPKLKAVDDLLRKQRVQLGNRVAASTKEGILSVIKEVRKGGSVGIPADPEPAESAGIFVPFCGTMALTSKFVPNMLAGGKAVGVFLHAMRLPDGSGYKVVLEAAPEAMYSTDTETSAAAMSKVVEKYVRAYPSQYMWTMKRFKKRPPGEARWY and the molecular was32651Da.Thirdly, MTT assay and trypan blue exclusion experiment were used to study the effect of WGWSP11on MDA-MB231cell viability and death rate respectively. The IC50values ofMTT after24,48and72h were respectively76.35,22.33and14.41μg/mL, and the IC50values of trypan blue exclusion after24,48and72h were respectively100.23,23.53and16.37μg/mL. The results indicated that the effects of WGWSP11on MDA-MB231cells wereconcentration-and time-dependent, and especially time had more significant impact. Typicalapoptotic morphology was observed by phase-contrasted microscopy, fluorescencemicroscope staining with Hoechst33258and scanning electron microscope. The cytotoxicityof WGWSP11on embryonic kidney cell HEK293was very weak.Finally, the preliminary mechanism was investigated in vitro by flow cytometry andDNA electrophoresis. Firstly, the apoptosis rate was determined by translocation ofphosphatidylserine to the cell surface using an Annexin V-FITC apoptosis detection, afterbeing treated with WGWSP11. As a result the early apoptosis rates after24h were6.55%,13.51%and18.25%, and late apoptosis rates after48h were69.29%,70.77%and90.36%,which revealed that more MDA-MB231cells were at late apoptosis after48h. Second, cellcycle distribution revealed by PI staning showed that WGWSP11arrested MDA-MB231cellsat G0/G1. Thirdly, the result of determination of mitochondrial membrane potential showedthat the ratio of red fluorescence to green fluorescence decreased from2.42to1.02afterMDA-MB231cells were treated with the WGWSP11after24h, and this downward trenddidn’t enhance with the increase of time. In addition, significant feature of apoptosis wasobserved by DNA electrophoresis technology, which indicated that the DNA was cracked intofragments when MDA-MB231cell was treated with WGWSP11. The results showed thatWGWSP11arrested MDA-MB231cell cycle at G0/G1, induced DNA specific cutting,decreased mitochondrial membrane potential, and it caused the release of cytochrome c and itis Caspase-dependent.