The Sanguinarine Induced Mice Colon Cancer Cell Apoptosis And Related Signaling Pathway Research

ObjectiveSanguinarine is the main chemical components of chinese medicine which comes from the Macleaya,the Eomecon and other aquatic plants.Over the past few years,through the study of sanguinarine discoverying with the value of development and utilization,which has significant anti-tumor effect.In this experiment,we have explored the CT26·WT colon cancer cells which from the inhibition of tumor cell proliferation in vitro study and tumor cell apoptosis and cell cycle which was measured after administration of tumor cells and other aspects of sanguinarine antitumor effects.We have tested the research object of a tumor-burdened mouse model in vivo to explore the sanguinarine antitumor mechanism.Our study includes the quality of survival for mice,the tumor inhibition rate,transplanted tumor cell apoptosis and cycle,angiogenesis and so on.That will provide the necessary scientific theory basis for the clinical application of sanguinarine to treat cancer.Methods and Results1.The extraction and separation of SanguinarineTake the Macleaya extract powder,While using the suitable 95%ethanolof suitable amount to extract and bath 60 minutes of 70?.Filtering the extraction,and allowing to cool.Then extracting with concentrated sulfuric acid solution which is adjusted to about PH=2,standing and separateing out the precipitate.Filtering the precipitate and vacuum drying.This is the alkaloids sulfate of Macleaya.Bathing with ammonia and chloroform refluxes 60 minutes of 60? to extract the alkaloids.Filtering the residue and collecting the filtrate which is concentrated.The concentrate is to expand on silica gel column chromatography which particle sizehas from 100 to 200 mesh.Using the ethanol elutes the orange yellow strips and concentrates the eluate which under the reduced pressure condition.Placing the concentrate at 4? to crystallize and dissolving the crystals in anhydrous methanol.Then obtaining the sanguinarine monomer whose the purity over 95%after recrystallizing the crystals.2.The study of sanguinarine on CT26·WT colon cancer cells of miceBy MTT method to determine its inhibition rate of CT26·WT cells crossing different concentrations of sanguinarine monomer to CT26·WT cells,and maps the inhibition growth curve of the tumor cell.Then calculates the IC50(50%inhibitory concentration).So we obtained the IC50 of 24h,48h,72h IC50 are 1.78?g/mL,1.87 ?g/mL,1.71 ? g/mL.The Annexin V-FITC/PI double staining method and PI staining method are to detect the effect of apoptosis and cycle of sanguinarine acting on CT26·WT cell.That showes that sanguinarine could induce apoptosis to CT26·WT cells and arrest corresponding the cell cycle in vitro to inhibit the proliferation of tumor cell.3.The preliminary study of mechanism of sanguinarine on a tumor-burdened mouseWe have found that the sanguinarine has anti-tumor effect in vivo for animal tumors through the establishment of a tumor-burdened mouse.We also use the Western-Blot method to detect the tumor tissue's protein expression which include ATM,Chk-2,P53,Bcl-2 and Caspase-3.The results show that the concentration of 10mg/kg,20mg/kg and 40mg/kg of Sanguinarine have better effects than the other concentration that can significantly inhibit tumor growth and improve survival of mice.According to the experimental datas,we find that there is a positive correlation between the therapeutic effect and the concentration of sanguinarine in BALB/C mice.Sanguinarine by inducing apoptosis in CT26·WT tumor cells and inhibition of tumor cell cycle to arrest generated effects.ConclusionAccording the studies in vitro and in vivo for BALB/C mice we foundthat the sanguinarine can significantly inhibited CT26·WT tumor cells to grow and also can improve the survival and the quality of life for mice.A t the same time,Sanguinarine will down-regulating the expression of certa in genes and mutant oncogene-induced for CT26·WT cells,while raising leve is of certain tumor suppressor gene expression,which plays one of theimportant mechanisms role in tumor suppression.Sanguinarine can activate ATM,Chk-2,P53,Bcl-2,Caspase-3 of signaling pathway.Thereby enhancing the effect of repairing the gene mutations for mice in vivo and accelerating cancer cell death process,then play a relevant role in the anti-tumor.