Experimental Study On Mechanisms Of Cortex Ailanthi Extracts In Inhibiting Archenteric Tumour

Introduction:Cortex Ailanthi Ailanthus altissima (Mill.)Swingle is a herb that has been found to have a wide range of functions, such as clearing away heat and eliminating dampness, astringent for spermatorrhea and excessive vaginal discharge, relieving diarrhea, arresting bleeding and insecticide. It can be used in the treatment of anti-inflammatory, leukorrhea with reddish discharge, hot and humid celiac diarrhea, bloody stools and uterine bleeding. In addition to the traditional efficacy, modem research found that Cortex Ailanthi also has the effect of anti-tuberculosis, anti-EBV, antiamebic, antimalarial and antitumor. Especially the properties of anti-cancer contains bitter principles known as to treat cerical cancer, colon and rectum cancer. In the present study, Cortex Ailanthi plants were sequentially extracted by water, petroleum ether (PE), ethyl acetate (EA) and each extract was examined for bioactivity by MTT assay in cancer cell lines to identify the most active fraction and the most sensitive cell line. The effect of the active site on apoptosis was determinded by ffow cytometry. Using Real-time PCR, we investigated the molecular mechanism by the active site in the sensitive cell line.Objective:The objectives of the present investigation were to determine whether Cortex Ailanthi extract has efficacy in suppressing proliferation of gastrointestinal tract cancer cell lines, to identify novel targets for Cortex Ailanthi extract in cancer cells and to explor the molecular mechanism that Cortex Ailanthi extract inhibits cancer cell growth in vitro.Methods:1. Plant extracts preparation:Cortex Ailanthi was extracted with water twice, using10volumes of water for1h each time and collection of the distillate. By vacuum concentration, we got water extrat35ml. Reserved sample5ml, the rest added ethanol containing alcohol volume was75%. Placed overnight and got84ml alcohol solution by pump filter. Reserved sample8ml, recovered ethanol from the rest of the solution. The solution was further partitioned with equal volume of petroleum ether (PE) and divided into PE and H2O fractions through liquid-liquid partition. The H2O fraction was further partitioned with equal volume of ethyl acetate (EA) and divided into EA and H2O subfractions. The water extract, the petroleum ether (PE) extract and ethyl acetate (EA) extract were concentrated by evaporator and then got the water extract, PE extract and EA extract.2. Assessment of cell growth inhibition by MTT assay:MTT was used to detect which extract (water extract、PE extract and EA extract) exhibited the highest degree of cytotoxicity to various cancer cell lines. 3. The EA subfraction was examined for bioactivity by MTT assay in MGC-803cells.4. The EA subfraction was identified by TLC in order to detect its composition roughly.5. Flow cytometry analysis of apoptosis:Flow cytometry were used to detect apoptosis in various cells treated with different doses of EA extract.6. Real-Time PCR was used to detect the expression of Ras/MAPK signal transduction pathway related genes after cell were treated with EA extract.Result:1. To determine the effect of three Cortex Ailanthi extract (water extract, PE extract and EA extract) on the proliferation of various cancer cell lines. The MTT assay results showed that EA extract exhibited the highest degree of cytotoxicity than others. Our results also showed that MGC-803is the cell line most sensitive to EA extract-induced cytotoxicity (IC50=63μg/ml) and showed a dose-dependent relationship.2. To analysis EA extract by TLC. The TLC result showed that EA extract mainly contains quassinoides.3. The variations of apoptosis upon treatment of various cells with different concentrations of EA extract were investigated by flow cytometry. We observed significantly Bel-7402and MGC-803cell following treatment with EA extract had different degrees of apoptosis. Apoptosis effect inducing by EA extract in MGC-803cells for48h was the strongest and the percentage of apoptotic cells was increased dose-dependently at (83.15±2.51)%.4. The expression of Ras/MAPK signal transduction pathway related genes in MGC-803cells treated with EA extract(1mg/ml) for6h by Real-time PCR. Results showed that treatment of MGC-803cells with EA extract significantly reduced the expression of SOS1, HRAS, MEK2, Raf-1, MEK1, whereas no obvious alterations of MAPK1and Grb2level were observed.Conclusion:In this study, Cortex Ailanthi plants were sequentially extracted by water, petroleum ether (PE), ethyl acetate (EA) and each extract was examined for bioactivity by MTT assay in cancer cells. Our results showed that the EA extract exhibited the highest degree of cytotoxicity to gastric carcinoma MGC-803cells. Further investigations revealed that treatment of EA extract mainly induced significantly apoptosis in MGC-803cells. EA extract treatment in MGC-803cells resulted in significant reduction of SOS1, HRAS, MEK.2, Raf-1and MEK1to control level.