A Study On Antitumor Effect And Mechanisms Of Pulsatilla Chinensis Saponins

Cancer, one of the most lethal diseases, is serious harmful to people’s lives. It’s very necessary to find out the effective prevention and treatment for the incidence increased year by year. Chemotherapeutics is one of the main clinical therapies of tumor, but chemotherapy drugs not only affect the target tumor cells, but also involved in normal cells. The serious side effects, e.g. immune hypo function, bone marrow suppression, organ damage and the multidrug resistantance, which greatly restricted the chemotherapy drug application. Traditional Chinese medicine has the characteristics of multiple components and multiple targets, can act on tumor different segments. Besides, low toxicity, enhancing the body immunity and antagonizing drug-resistance make it become a hot study of antitumor drug in recent years.Heat-clearing and detoxicating drug, tonification drug, huoxuehuayu medicine, lishuishenshijianpi medicine, these Chinese medicines often used to treat tumor, huoxuehuayu medicine is the most important in percentage. Pulsatilla Chinensis is a huoxuehuayu medicine. Anemone pulsatilla saponin extract from Pulsatilla Chinensis, previous study showed that, antitumor effective part in vitro was identified through experiments. Then main components were isolated and purified from Anemone pulsatilla saponin to investigate the structure-activity relationship and the anti-tumor activity in vitro. At present, B3, B7, B9and B11were identified which have the antitumor activity. Based on those results, we study the pharmacodynamic action and antitumor mechanism of satilla Chinensis, to provide a sound theoretical basis for the new medicament research and the clinical application. ObjectiveBased on the previous study, we carry out the pharmacodynamic study and the antitumor mechanism research of Anemone pulsatilla saponin, the effective components of B3, B7, from inducing tumor cells apoptosis, autophagy and energy metabolism. To clarity whether Anemone pulsatilla saponin’multi targets synergy regulation, the mechanism research of the multiple components (B3,B7) is carried out for annotation the antitumor effect of multi components, multi pathway, multi mechanism of Pulsatilla saponins, to provide a sound theoretical basis for the new medicament research and the clinical application.MethodsPart One Establish of anti-tumor active fraction and active constituent in Pulsatilla chinensis (Bunge) Regel.Culture huamn hepatocellular carcinoma BeI-7402cells and huamn glioma SHG-44cells in vitro; Establish H22hepatocellular carcinoma model in vivo to determin the antitumor effect of Pulsatilla saponins in vitro and in vivo,Culture huamn lung cancer A549cells, human hepatocellular carcinoma SMMC-7721, human colorectal cancer cell line HT-29cells in vitro; Establish H22hepatocellular carcinoma model in vivo to evaluate the antitumor effect of B3, B7, B9,B11.Part Two Studies on anti-tumor therapeutic effects of Pulsatilla saponin1Studies on anti-tumor therapeutic effects of Pulsatilla saponins in vivoCulture12kinds of tumor cell lines, BGC-823、SGC-7901、NCI-H460、 A549、SMMC-7721、Bel-7402、HT-29、HCT116、U251、SK-OV-3、PC-3and K562. Using MTT assay to detect the suppression rate of pulsatilla saponins (%) and calculate the half suppression rate IC50(μg/ml) 2Anti-tumor effects of Pulsatilla saponins on H22/CT26.WT xenograft model Colon cancer cells CT26.WT and hepatoma H22cells are inoculated to the right armpits of BALB/c mice and KM mice to establish mice bearing tumor model of mice colon cancer CT26.WT, liver cancer H22. Give corresponding drugs14days later, weigh the tumor and calculate the inhibition rate, and observe the inhibiting effect of pulchinenoside to all kinds of tumors. Weigh the spleen and thymus, then to calculate the organ coefficient of every group. Collect blood to examine the situation of blood cells with full automated hematology analyzer to detect the influence to blood cells of pulsatilla saponins.3. Anti-tumor effects of Pulsatilla saponins on Bel-7402/HT29xenograft model in nude miceRespectively inoculate human colon cancer HT29cells and human hepatocellular carcinoma Bel-7402cells to be transplantation tumor in nude mice, then cultured tumor block more than2generations, the trocar were inoculated in nude mice subcutaneous, establishment of human colon cancer xenograft tumor model/hepatocellular carcinoma nude mice model. Administration of24days, tumor volume measurement,1/3d, after given the last drug, tumors were weighed to calculate the tumor inhibition rate (%), to observe the effect of the transplantation tumor of Pulsatilla saponin; Weigh the spleen and thymus, then to calculate the organ coefficient of every group. Collect blood to examine the situation of blood cells with full automated hematology analyzer to detect the influence to blood cells of Anemone pulsatilla saponin.Part Three Studies on anti-tumor mechanism of Pulsatilla saponins1.Effect of inducing apoptosis of Pulsatilla saponinsPulsatilla Saponin intervene human colon cancer HT-29cells for48hr in vitro, using DAPI staining, apoptosis morphology of tumor cells was observed under the inverted fluorescence microscope; Using AnnexinV/PI double staining to observe the effect of Pulsatilla Saponin on HT-29cells apoptosis by the flow cytometry.2.fect of inducing autopathy of Pulsatilla saponinsPulsatilla Saponin intervene human colon cancer HT-29cells for48hr in vitro, immunofluorescence staining was used to observe the expression and distribution of autophagy marker, LC3; Western blotting was used to detected the expression of LC3to evaluate the effect of Pulsatilla Saponin on autophagy.3. Effect of regulating energy metabolism of Pulsatilla saponinsEstablish human hepatocellular carcinoma Bel-7402nude mice xenograft tumor model, after Pulsatilla Saponin intervention, to observe the change of Pulsatilla Saponin on the level of lactate and ATP in tumor tissues using biochemical assays; to observe the effect of Pulsatilla Saponin on hexokinase II (HK II), phosphofructokinase (PFK), pyruvate kinase (PK) and succinate dehydrogenase in tumor tissues by colorimetric method and ELISA assay.4The anti-tumor mechanism of the effective components of Pulsatilla SaponinsB3/B7intervene human colon cancer HT-29cells for48hr in vitro, using DAPI staining, apoptosis morphology of tumor cells was observed under the inverted fluorescence microscope; Using AnnexinV/PI double staining to observe the effect of B3/B7on HT-29cells apoptosis by the flow cytometry. immunofluorescence staining was used to observe the expression and distribution of autophagy marker, LC3; Western blotting was used to detecte the expression of LC3to evaluate the effect of B3/B7on autophagy. To observe the effect of B3, B7on lactate and glucose content the supernatant of cultured detected by biochemical assay. 5The molecular mechanism of antitumor effect of Pulsatilla Saponins and its effective components based on Akt pathwayPulsatilla Saponin, B3and B7intervene human colon cancer HT-29cells for48hr in vitro, using PCR chip, to observe the effect of Pulsatilla Saponin, B3and B7on the target genes, which are the AKT downstream related with apoptosis, autophagy and energy metabolism pathway members FOXO1, Bim, Caspase9, Bcl-2, Bax, Caspase3, Atgl, Atgl3.Beclinl, PI3K, III, P70S6K, Bad, GSK-3β, GLUT1, HIFa, in order to clarify the role of characteristics of Pulsatilla Saponins of multiple components, multiple targetsResultPart One Establish of anti-tumor active fraction and active constituent in Pulsatilla chinensis (Bunge) Regel.The results showed that Pulsatilla saponins extract isolated from Pulsatilla chinensis (Bunge) Regel. had good anti-tumor effects in vivo and vitro, IC50value is5.12μg/ml for human hepatoma Bel-7402cells,2.26μg/ml for hepatoma SMMC-7721cells,2.03μg/ml for human HT-29colon carcinoma cells,3.24μg/ml for human HCT-116colon carcinoma cells, showed good antitumor activity in vitro; it also had a significant anti-tumor activity in vivo with tumor inhibition rate of67.5%on H22hepatocellular carcinoma xenograft model.Further efficacy screening of active constituents isolated from Pulsatilla saponins showed, B3, B7, B9, B11has good anti-tumor activity in vitro,espescially B3, B7; some of the constituents show the same anti-tumor activity with Pulsatilla saponins.Part Two Studies on anti-tumor therapeutic effects of Pulsatilla saponins1.Studies on anti-tumor therapeutic effects of Pulsatilla saponins in vivo Anti-tumor experiments in vitro showed that Pulsatilla saponins had good inhibitory effect on12subjects tumor cell lines, at the concentration of25μg/ml, the inhibition rates were over96%at a good dose-effect manner. Average of IC50value ranged3.44to9.91μg/ml, had significant antitumor activity in vitro.2.Anti-tumor effects of Pulsatilla saponins on H22/CT26.WT xenograft modelThe tumor inhibition rate of Pulsatilla saponins at the dose of high, medium and low dose (300,200,100mg/kg) are74.37%,61.59%and74.37%respectively on H22hepatocellular carcinoma xenograft model;67.9%,51.35%,22.03%on CT26.WT colon carcinoma xenograft model.It had certain influece on animal body weight and spleen,but had no side effect on WBC, RBC, hemoglobin, platelet etal.3.Anti-tumor effects of Pulsatilla saponins on BeI-7402/HT29xenograft model in nude miceThe tumor inhibition rate of Pulsatilla saponins at the dose of high, medium and low dose (300,200,100mg/kg) are42.8%,31.5%and14.9%respectively on Bel-7402hepatocellular carcinoma xenograft model;30.9%,23.5%and17.8%on HT29colon carcinoma xenograft model. It had certain influece on animal body weight and spleen,but had no side effect on WBC, RBC, hemoglobin, platelet etal.Part Three Studies on anti-tumor mechanism of Pulsatilla saponins1.Effect of inducing apoptosis of Pulsatilla saponinsThe number of apoptotic HT29cells increased with the dose of Pulsatilla saponin after treatment for48hr,apoptotic HT29cells displayed around and shrunken cell body, the apoptotic rate was increaed at a dose-effect manner after treatment with Pulsatilla saponin,and had significant difference compared with control group.This suggested that Pulsatilla saponin could induce apoptosis of HT29cells.2.Effect of inducing autophagy of Pulsatilla saponinsLC3protein of HT29cells in control group was homogeneous distribution in cytoplasm,but in Pulsatilla saponins group,there was activation and degradation of autophagy protein LC3, and autophagosome was found; Western blotting assay showed that after treatment with Pulsatilla saponins, type cytoplasm LC3(LC3-I) degraded into type (autophagy) film (LC3-Ⅱ), indicated that Pulsatilla saponins can induce HT29cell autophagy.3. Effect of regulating energy metabolism of Pulsatilla saponinsPulsatilla saponins could decrease the content of lactic acid and ATP.It also could decrease the levels of glycolytic eruzymes PFK,HK-Ⅱ,PK,and increase the levels of SDH that was the most enzymes during tricarboxylic acid cycle. This indicated that Pulsatilla saponins can regulate energy metabolism of tumor cells.4.Studies on anti-tumor mechanism of active constituent in Pulsatilla saponinsCompared with control group,B3/B7can induce tumor cell apoptosis, in addition to the B3group at low doses of2.5μg/ml, cell apoptosis rate of the remaining groups had significant difference compared with the control group; B3and B7at each dosage were expressing LC3-Ⅱ, and increased with the rising of concentration; B3, B7can reduce HT29cell glucose uptake, and the formation of lactic acid.5.The molecular mechanism of antitumor effect of Pulsatilla Saponins and its effective components based on Akt pathwayCompared with control group, Pulsatilla saponins could reduce Akt,Caspase9, Caspase3, GLUT1gene expression significantly (P<0.05,P<0.001), significantly increase Atgl gene expression (P<0.01);B3could decrease Akt,Caspase9,Atgl3gene expression significantly (P<0.05,P<0.01); B7could decrease Akt,Caspase9, Caspase3, Atgl3,GLUT1gene expression significantly (P<0.05, P<0.01), and increase Beclinl gene expression significantly (P<0.05). Compared with Pulsatilla saponins group, Caspase9,Caspase3,GLUTlgene expression in B3group significantly raised (P<0.05-P<0.05), Atgl gene expression significantly lowered (P<0.05); Caspase9, Caspase3gene expression in B7group was obviously higher (P<0.05, P<0.01), Atgl3, Atgl gene expression significantly lowered (P<0.05).Conclusion1. Pulsatilla saponins have good anti-tumor activity in vivo and in vitro, Average of IC50value ranged3.44to9.91μg/ml; it showed good inhibitory effect on H22liver cancer, CT26. WT colon cancer xenograft model in mice, and on Bel-7402, HT29xenograft model in nude mice, and had no obvious side effect on leukocyte.2. Pulsatilla saponins inhibited tumor growth by regulating the Akt pathway, thus inducing tumor cells apoptosis, autophagy, regulating tumor cells energy metabolism; And its effective components of B3, B7alos could induce tumor cells apoptosis, autophagy, regulating tumor cell energy metabolism, which indicated that anti-tumor effects of Pulsatilla saponins were realized by its B3, B7components working together.