Experimental Research Of Rhizoma Dioscoreae Bulbiferae Inhibition Of Growth And Metastasis In Gastric Cancer

Gastric cancer is one of common malignant tumors in worldwide. The global new cases of primary gastric cancer are990000in2008, accounting for8%of all new cancer cases. And740000were died, accounting for10%of all cases of death. At present, GC tumor mortality is accounted for the second, only followed by lung cancer in the world. According to epidemiology statistics, China’s gastric cancer incidence rate ranked third in2000, and has risen to second from2004to2007. T he total incidence shows a steady trend. According to the China health statistics annual report in2010, the annual mortality rate of gastric cancer in China was23.10/10million, which was more than2times of the world average.Gastric cancer has become a major disease that threatens the people’s health, and hinders economic and social development. At present the major treatment of gastric cancer is surgical operation. The patients with early gastric cancer postoperative5years survival rate is high, which may reach above90%. But the vast majority of gastric cancer is advanced at the time of diagnosis, and has lost the chance of radical operation.5year survival rate of it’s is only11%-40%. Western medicine for advanced gastric cancer is radical or palliative operation, radiotherapy and chemotherapy immunotherapy treatment. However, western medicine curative effect of worldwide advanced gastric cancer is not ideal. Thus treatment of traditional Chinese medicine has a considerable role. Therefore, it has strong clinical and practical significance to further look for strong specificity and high efficiency low toxicity of antitumor drugs. In recent years, traditional Chinese medicine in the prevention and treatment of gastric cancer plays a more prominent role. And the studies in vitro have confirmed that Chinese medicine anti-tumor effect is exact. Clinical practice shows that separate application of traditional Chinese medicine or Chinese medicine compound treatment of gastric cancer has curative effect, at the same time, that can reduce the side effects of radiation and chemotherapy. Rhizoma dioscoreae bulbiferae is the tuber of potato plants Dioscorea bulbifera1. It is cold and bitter taste,and it has the role of cooling blood, eliminating gall, sending fire and detoxification. Rhizoma dioscoreae bulbiferae is widely used in clinical. It mainly treat a variety of disorders. Past literature reported that Chinese medicine rhizoma dioscoreae bulbiferae has powerful antitumor effect. Clinical reports that using huangbai stomach soup containing rhizoma dioscoreae bulbiferae to treat gastric cancer, cardia cancer and esophagus cancer have curative effect. There have reports that rhizoma dioscoreae bulbiferae was used to treat liver cancer, breast cancer, cervical cancer, primary bronchial lung cancer, lymphoma, etc. It also has certain curative effect. Related studies have shown that Chinese medicine anti-tumor mechanisms include inhibiting the expression of proto-oncogenes, promoting the expression of tumor suppressor genes, regulating of cancerous tissue surrounding environment, inducing cancer cell apoptosis, reducing the ability of gastric cancer cell invasion and metastasis. But the concrete mechanism of rhizoma dioscoreae bulbiferae is not clear for the treatment of gastric cancer. To study the above problems, this study proposed by observing the effect of malignant biological behavior of dioscoreae bulbiferae on cultured in vitro. And preliminary explore the molecular mechanism of its action and establish the animal model of nude mouse stomach subcutaneous transplantation tumor dioscoreae bulbiferae. Then we observe the effect of rhizoma dioscoreae bulbiferae on a tumor-burdened rat growth inhibition. This study of rhizoma dioscoreae bulbiferae can provide scientific theoretical and experimental basis for treatment of cancer of the stomach, and can enrich anti-tumor theory at the same time. It is of great significance to promote the traditional Chinese medicine clinical transformation.Objective1To observe of the effect of redox ability, proliferation and metastasis by dioscorea bulbifera on gastric cancer SGC-7901cells in vitro, and to investigate the molecular mechanism of dioscoreae bulbiferae inhibition of gastric cancer growth and metastasis.2To observe the effect of the anti-tumor effect of dioscorea bulbifera by investigating the tumor growth in animal model of gastric cancer.Method1Gastric cancer cell was cultured in vitro, and dioscorea bulbifera was extracted by ethanol to make of different concentration solution.2We use the spectrophotometric to detect the clearance rate of hydroxyl radical and DPPH radical of alcohol extraction of dioscorea bulbifera of different concentration on gastric cancer cell.3We use ABTS rapid test to detect the antioxidant activity of extracts of different concentrations of alcohol extraction of dioscorea bulbifera on gastric cancer cells and reduction ability.4We use MTT method to detect the effect of proliferation of different concentrations of alcohol extraction of dioscorea bulbifera on gastric cancer cells.5We use the flow cytometry to detect the effect of different concentrations on the apoptosis of gastric cancer cells.6We use the Transwell invasion to detect the effect of different concentrations of Dioscorea bulbifera alcohol effect on the invasion of gastric cancer cells.7We established the gastric carcinoma transplanted subcutaneously in nude mice and observed the effects of ethanol extract of Dioscorea bulbifera L on tumor growth.8We use the ELISA to detect the expression of the inflammatory cytokines in peripheral blood of gastric cancer in nude mice. We observed the effects of inflammatory cytokine concentrations of ethanol extract of Dioscorea bulbifera L.All data used SPSS13statistical software. The significant level of α=0.05, with P＜0.05as the difference has statistical significance. Result1SpectrophotometryDifferent concentrations of rhizoma dioscoreae bulbiferae ethanol extracts has certain hydroxyl radicals and DPPH free radical clearance. In the three kinds of extracts, the DPPH radical scavenging capacity with concentration of2mg/ml,70%ethanol extract was strongest. It’s clearance ratewhich was55.2%. The OH radical scavenging capacity of80%ethanol extract was the highest. It’s clearance rate was51.2%. And the OH radical scavenging capacity of70%and90%ethanol extracts were38.7%and39.6%respectively.2Reducing power testRhizoma dioscoreae bulbiferae ethanol extracts have stronger reducing power. The reducing power of70%ethanol extract was28.1μmol Fe2+, The reducing power of80%ethanol extract was equival to49.3μmol Fe2+per1g, the reducing power of90%rhizoma dioscoreae bulbiferae ethanol extracts was equival to35.2μmol Fe2+per1g.80%ethanol extract has the strongest reducingpower.3Rapid detection of ABTSRhizoma dioscoreae bulbiferae ethanol extracts had stronger antioxidant capacity. The removal of the80%ethanol extract was highest, which was close to72%. The clearance rate in70%and90%ethanol extracts of was55%and45%respectively. And the total antioxidant capacity increased gradually with the time going and the concentration increasing. When the lowest concentration was0.2mg/ml, the clearance rate was8%. When the highest concentration was2mg/ml, the clearance rate was52%. When4min,7min and12min, the concentration was0.2mg/m,0.4mg/m and1.0mg/ml. The clearance rate has risen steadily. When the concentrations were1.4mg/ml,1.8mg/ml and2mg/ml, the changes of free radical clearance were not obvious, that were generally between55%and67%.4Detection cell proliferation by MTT methodDifferent concentrations of rhizoma dioscoreae bulbiferae ethanol extracts solution could inhibit gastric cancer SGC-7901cells’ proliferation.70%rhizoma dioscoreae bulbiferae ethanol extracts solution with0.5mg/ml1mg/ml and2mg/ml could inhibit gastric cancer SGC-7901cell proliferation rate, there are34.5%,41.2%and34.5%respectively. The cell proliferation rate in inhibits gastric cancer SGC-7901of80%rhizoma dioscoreae bulbiferae ethanol extracts solution were0.5mg/ml、1mg/ml、2mg/ml, they were41.3%、55.8%and62.3%respectively. In90%rhizoma dioscoreae bulbiferae ethanol extracts solution with0.5mg/m、1mg/m、2mg/ml, the cell proliferation rate to inhibit gastric cancer SGC-7901were28.7%,32.4%and28.7%respectively. The inhibition rate of80%rhizoma dioscoreae bulbiferae ethanol extracts had the highest of62.3%.5Flow cytometry instrument to detect cell apoptosisAfter culture48h, rhizoma dioscoreae bulbiferae ethanol extracts could obviously promote the SGC-7901cells apoptosis, and the control group only had a small amount of SGC-7901cells apoptosis.The cell apoptosis rate in70%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、lmg/m and2mg/ml were rised from the blank control group (3.81±1.56)%respectively up to (5.36±1.85)%,(10.31±2.46)%(P＜0.05, and (18.54±3.26)%(P＜0.01);0.5,1and2mg/ml airpotato yam80%ethanol extract of SGC-7901cell apoptosis rate respectively (6.62±1.15)%,(14.27±2.28)%(P＜0.05) and (25.07±4.21)%(P＜0.01), and a certain concentration dependence.0.5,1and2mg/ml airpotato yam90%ethanol extract processing of SGC-7901cell apoptosis rate (5.65±1.63)%,respectively(11.81±2.75)%(P＜0.05) and (20.32±3.54)%(P＜0.01).6Transwell invasive testing Human gastric cancer SGC-7901cells were cultured24hours respectively with80%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、1mg/m and2mg/ml. Compared with the blank control group, the cell invasion number showed a trend of decrease. We count the cells number under through the room at high magnification, the control group was94.36±9.51,80%rhizoma dioscoreae bulbiferae ethanol extracts with0.5mg/ml were67.52±7.48.80%rhizoma dioscoreae bulbiferae ethanol extracts with1mg/ml were51.24±6.51.80%rhizoma dioscoreae bulbiferae ethanol extracts with2mg/ml were38.59±5.43. Compared with control group,80%rhizoma dioscoreae bulbiferae ethanol extracts of SGC-7901with0.5mg/m、1mg/m and2mg/ml can inhibit gastric cancer cell line (P＜0.05). There was a dose-response relationship.7Subcutaneous transplantation tumor model of human gastric cancer in nude miceThe tumors were implanted subcutaneously in nude mice, up to seventh days, the formation of local measurable tumor, model success rate of100%; subcutaneous tumor growth is slow, the appearance of spherical or hemispherical, early active, gradually hardens, tumor size is not a group, the observation period nude mice showed no obvious weight loss and weight loss.8Effect of dioscorea bulbifera L alcohol extracts on tumor volumeAfter the end of treatment, tumor volume of high dose group is smaller than that of the model group (P＜0.05), the volume of5-FU group and low dose group and model group showed no statistical difference (P＞0.05), compared with5-FU group, high-dose group and low dose group, there were no significant differences (P＞0.05),5-FU group and low, high dose group three inhibition on the tumor volume was respectively25%,17.86%and39.29%.9Inhibition of tumor weight rate by dioscorea bulbifera alcohol extractCompared with the model group,5-FU group and high dose group of dioscorea bulbifera nude mice transplanted tumor mass was significantly decreased (P＜0.05), between different dose groups of Dioscorea bulbifera1tumor weight had no significant difference (P＞0.05), low dose of group tumor mass compared with5-FU group, the difference had no statistical significance (P>0.05),5-FU group and Dioscorea bulbifera low, high dose group of nude mice tumor mass was respectively37.5%,25%and43.75%.10IL-8level of dioscorea bulbifera alcohol extract on tumor peripheral blood serumCompared with the model group, serum IL-8levels decreased significantly in5-FU group and dioscorea bulbifera group (P<0.05); the serum IL-8levels in high dose group were significantly lower than those of low dose group (P＜0.05).11sICAM-1level of serum level of dioscorea bulbifera alcohol extract on tumor peripheral blood serumCompared with the model group, serum sICAM-1levels decreased significantly in5-FU group and dioscorea bulbifera group(P<0.05); serum sIC AM-1levels in high dose group were significantly lower than those of the low dose (P＜0.05). Conclusion1Rhizoma dioscoreae bulbiferae alcohol extract has the certain ability of free radicals scavenging,reduction and antioxidant on human gastric cancer cell line.2Rhizoma dioscoreae bulbiferae alcohol extract can inhibit proliferation and induced apoptosis ofhuman gastric cancer SGC-7901cell.3Rhizoma dioscoreae bulbiferae alcohol extract can reduce the invasiveness of human gastric cancer SGC-7901cell line.4Rhizoma dioscoreae bulbiferae alcohol extract can inhibit the growth of subcutaneous xenograf in gastric cancernude mice, and its possible mechanism of invasion and metastasis was related to downregulation of inflammatory factors (IL-8and sICAM-1).