ComParative Proteomics Studies Of RicinToxin-induced Apoptosis In Hutu-80cells

The ricin toxin (RT), derived from the seeds of the castor oil plant Ricinus communis, is a high performance of Ribosome-inactivating protein (RIP) protein, Which is comprised of two glycoprotein chains of roughly equal size, chains A(RT A-chain, RTA) with N-glycosidase activity that it leading to protein synthesis inhibition, and cell death and chanins B with lectin activity that it can identify and combination containing structure of D-galactose on cell surface receptors and helping a chain A into the cellular. After a single RT molecule into the cell it can make whole cell protein synthesis stop eventually cause cell death. This study we analyzed whole protein differential expression through HuTu-80cell by comparative proteomic research tools, which provided a new test window for the mechanism of action of ricin the HuTu-80cells.1. This study we first adopt ammonium sulfate method. getting the crude poison ricin from castor beans and purified ricin through Sepharose4B affinity chromatography and S-200column chromatography, added β-mercaptoethanol treatment, we found that ricin obtained RTA of molecular weight was28KDa, while RTB of ricin obtained molecular weight was32KDa, finally get electrophoresis with pure ricin were carried out IC50analysis in our experiment. SvS-PAGE results showed that we extracted for electrophoresis pure ricin, extracted ricin on Hutu-80cells12hours IC50was200ng/ml, the cell morphology changed.2. The cell model of of RT-induced apoptosis in a human Hutu-80cell line were studied. To shed light on the mechanism of action of RT at the cellular level,we examined cell growth, apoptosis, changes of mitochondrialmembrane potential (MMP) and cytochrome C translocation in HuTu-80cells by exposing these cells to RT for indicated times. The effect of RT on cell proliferation was measured by MTT, inner salt; MTT assay and apoptosis were measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes in MMP were monitored using flow cytometry. Western blot analysis was used to evaluate the release of mitochondrial cytochrome C. RT noticeably inhibited the proliferation of HuTu-80cells, and the half maximal inhibitory concentration dose was about200ng/ml. HuTu-80cells treated with RT showed typical characteristics of apoptosis rather than necrosis, including phosphatidylserine exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation However, when cells were treated with RT, the massive translocation of cytochrome C to the nucleus was evident. Our results indicate that RT-induced Hutu-80cell apoptosis. This results indicated that we have successfully constructed Hutu-80cell apoptosis model induced by RT exposure.3. When total protein at200ng/ml Ricin toxin treatment8h from normal and treated HuTu-80cells protein, we get1660differentially expressed proteins information quantitative analysis by iTRAQ isotope labeling differences proteins and mass spectrometry techniques. we will gain94differentially expressed proteins, that is p-value<0.05, which provides a reference for future studies of ricin the induction HuTu-80cell apoptosis mechanism.