Anti-tumor Activity Of The Total Glucosides Of Radix Paeoniae Rubra And The Underlying Mechanisms In S180 And K562

Objective: In China, cancer is one of most serious diseases at present and treatment options are limited. Herb medicines have been widely used as an alternative method but the underlying mechanisms remain largely unknown. In this study, we have investigated the anti-tumor effect and the molecular mechanisms of Total Glucosides of Radix Paeoniae Rubra (TGC) in order to establish the experimental systems for further studies of Traditional Chinese Medicine in cancer treatment, either as a primary therapy or in combination with other conventional interventions.Methods:①In vivo experiments using murine tumor models: KM mice were inoculated with syngenic tumor cell line S180 or H22. One day after tumor inoculation, mice were treated with TGC, cyclophosphamide(CY), combination of TGC and CY,or left untreated as control. The administration lasted 7 days and mice were sacrificed on day 8. Sera and tumor tissues were collected for further analyses.②In vitro analyses of anti-tumor effect by TGC on human tumor cell lines: Cultured cell lines including K562 (erythroleukmia), Hela (cervical carcinoma), A549 (lung carcinoma), Bel-7402 (hepatoma) and HCT-8 (colon carcinoma) were treated with TGC at different concentrations (0,25,50 and 100μg/ml). TGC has an important influence on induced tumor apoptotic mechanism of action by in vitro.③50 male KM mice inoculated with S180 cell strain were randomly divided into six groups: the normal group, control group, cyclophosphamide(CY)group, TGC group, combination of cyclophosphamide and TGC group(CY+TGC), therapy are respectively given to mice the second day. The administration last 7days and then sacrificed the mice. We have collect serum and strip the tumor tissue in order to determinate correlate index about induced tumor apoptosis and resist immune escape.Results:1. TGC has an anti-tumor effect in vivo. TGC treatment was more effective in S180 model, compared to H22 tumor, and a suppression rate of 42.81% was achieved. Further analysis by flow cytometer indicated that a great number of S180 cells were under apoptosis and tumor cells were blocked at the stage of S period.2. TGC inhibits the proliferation of human tumor cell lines in vitro. MTT assay indicated that K562 was the most sensitive line to TGC treatment among tumor cell lines tested. 45.9% of cultured K562 cells were inhibited when the concentration of TGC reached 50μg/ml. A hercalculation sensitivity is compared with IC50.the consequence manifestate that human erythroleukemia K452>human cervix carcinoma Hela>lung cancer A549>hepatoma Bel-7402>colon carcinoma HCT-8.3. TGC had markedly inhibited the proliferation on K562 cell in a time-dependent and dosage-dependent . K562 cell has been treated with TGC.then morphology change was observed under light microscope by Wright-Giemsa stain after 24 hours. TGC can inhibit G0/G1 period cells transfer to S period cell and increase apoptosis rate.the investigation has proved that TGC can urge K562 cell : evert membrane phosphatidyl serine,increase the level of Ca2+ and decrease mitochondrial membrane potential (MMP). Through real time PCR and WB experimental skill Cyto-Chrome C has been increased and caspase-3mRNA , caspase-9mRNA amplificated. Caspase-8mRNA has not any changed.TGC can regulate the expression of gene proteinum,such as decrease the expression levels of Bcl-2,Bcl-Xl and increase the expression levels of Bax in K562 cell.3.The total glucosides of radix paeoniae can inhibit tumor cell significantly through Depressing rates,morphous index,cell cycle.TGC can decrease the expression levels of Bcl-2,c-myc mRNA and increase the expression levels of Bax,P16 in S180 tumor tissue.4.TGC can inhibit the secrete of TGF-β1,increase the expression of MHC-Ⅰ,MHC-Ⅱprotein,increase the expression of MHC-ⅠmRNA.Conclusion: we have deeply investigated through statistics analyze and index relationship.the conclusion as follow:1.TGC can significantly inhibit S180 and K562 tumor cell through morphous index,MTT,cell cycle under a great quantity of experiment.2.TGC can induce K562 cell apoptosis in vitro .The potential mechanism of apoptosis might be related to evert membrane phosphatidyl serine and calcium overload. Ca2+ can open the mitochondrial permeability transition pore.so that mitochondrial outer membrane disrupt and△ψm descent or disappear.then respiratory chain shed couple,glutathione exhaust, reactiveoxygen species produce,protease activator release.such as Cyto-Chrome C set free and then in order activation caspase-9,caspase-3,evoke PARP degradation, the last revoke nucleolus DNA gragmentation a series of apoptosis reaction.when cell receive dead signal stimulus,the expression of anti-apoptosis gene bcl-2,Bcl-Xl is decreased,but promote apoptosis gene bax is increased, Bax protein can replaced emergence to increase mitochindria menbrane permeability. a lot of material can release in order to cell death.3. TGC may change the expression of associated protein Bcl-2,Bax,P16,c-myc. Bcl-2 protein oppose widl type p53 protein to retrain multi-factor which can induce cell apoptosis. It cantrol the dynamic balance between cell multiplication and apoptosis. Bax where is the surface of mitochondria destory the function of Bcl-2 protein.Both sides ratio effect the apoptotic proportion when cell receive post-stimulus. Tumor cell happen apoptosis co-induction that Bcl-2 protein step down and Bax protein step up.In a way , the transcriptional level of c-myc gene reflect the tissue and cell vegetative state. c-myc gene induce high express to impact cell cycle and promote transform cell phase.TGC inhibit proliferation tumor cell through raise up anti-oncogene P16 and down regulation c-myc. 4.TGC can inhibit the secrete of TGF-β1,increase the expression of MHC-Ⅰ,MHC-Ⅱprotein,increase the expression of MHC-ⅠmRNA. We can reveal expression of immune molecular on cell surface to strengthen specificity celluar reject tumor immunity response.The important measure restrain tumor cell tranfer and recure in internal.