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Objective: To investigate the effect of toosendanin (TSN) on the growth and invasion of gastric cancer cells BGC-823 by regulating circDLST expression. Methods: After gastric cancer cells BGC-823 were exposed to different con-centrations of TSN (0, 0.5, 1.0, and 2.0 μmol/L) for 24 h, quantitative PCR was used to detect the expression of circDLST. BGC-823 cells were transfected with the circDLST overexpression lentiviral vector or its control vector (CON), and then treated with 0.5 μmol/L TSN or PBS. So the cells were divided into circDLST+TSN group, CON+TSN group and CON+PBS group. The viability and invasive potential of BGC-823 cells were observed by MTT proliferation test and Transwell invasion assay. The subcutaneous transplanted tumor models were established by using circDLST-transfected cell line BGC-823 or the control cell line in nude mice, and then 200 μg/kg TSN or the same volume of PBS was injected intraperitoneally every day. So the mice were divided into circDLST+TSN group, CON+TSN group and CON+PBS group. Results: Compared with the control group (0 μmol/L), all 3 concentrations of TSN decreased the expression levels of circDLST in a concentration dependent manner (P<0.01). TSN could significantly reduce the cell viability, cell invasion and subcutaneous xenograft tumor growth (P=0.000), while circDLST overexpression reversed the inhibitory effect of TSN (P<0.01). Conclusion: TSN may inhibit the growth and invasion of gastric cancer cells BGC-823 by downregulating circDLST expression.
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OBJECTIVE: To study the effect of toosendanin (TSN)on the apoptosis of human ovarian cancer cell and to clarify the related mechanism. METHODS: CAVO-3 and A2870 cells were treated with toosendanin of different concentrations, and CCK-8 method was used to detect the cell survival rate, colorimetric method was used to measure the activities of Caspase-3 and Caspase-9, EdU method was used to determine the cell proliferation rate, and Western blot method was used to test the expressions of Bcl-2, Bax and Cyto-C protein. RESULTS: TSN could significantly inhibit the survival of CAVO-3 and A2870 cells with dose-(r=0.869 1, P<0.05) and time-(r=0.776 5, P<0.05) dependent manners. The survival rate of A2870 cell with TSN dropped significantly (P<0.05). The activities of Caspase-3 and Caspase-9 were significantly increased by TSN in CAVO-3 and A2870 cells, however, the inhibitors of Caspase-3 (z-DEVE-FMK) and Caspase-9 (z-LEHD-FMK) could reverse those effect of TSN (P<0.05) and also reverse the survival rates of CAVO-3 and A2870 cells (P<0.05). In addition, TSN could increase the expression of Bcl-2, Bax and Cyto-C protein in A2870 cells notably, which could be reversed mostly by Caspase-9 inhibitor (z-LEHD-FMK)(P<0.05). CONCLUSION: TSN could induce the apoptosis in human ovarian cancer cell through up-regulating the activities of Caspase-3 and Caspase-9, and then activates the mitochondrial pathway.
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Two new limonoids, 12-ethoxynimbolinins G and H (compounds 1 and 2), and one known compound, toosendanin (Chuanliansu) (compound 3), were isolated from the bark of Melia toosendan. Their structures were elucidated by spectroscopic analysis and X-ray techniques. The absolute configuration of toosendanin (3) was established by single-crystal X-ray diffraction. Compounds 1-3 were evaluated for their cytotoxicity against five tumor cell lines.
Subject(s)
Humans , Cell Line, Tumor , Cell Proliferation , Limonins , Melia , Chemistry , Molecular Structure , Plant Bark , Chemistry , Plant Extracts , Chemistry , Pharmacology , X-Ray DiffractionABSTRACT
Two new limonoids, 12-ethoxynimbolinins G and H (compounds 1 and 2), and one known compound, toosendanin (Chuanliansu) (compound 3), were isolated from the bark of Melia toosendan. Their structures were elucidated by spectroscopic analysis and X-ray techniques. The absolute configuration of toosendanin (3) was established by single-crystal X-ray diffraction. Compounds 1-3 were evaluated for their cytotoxicity against five tumor cell lines.
Subject(s)
Humans , Cell Line, Tumor , Cell Proliferation , Limonins , Melia , Chemistry , Molecular Structure , Plant Bark , Chemistry , Plant Extracts , Chemistry , Pharmacology , X-Ray DiffractionABSTRACT
AIM:To investigate the effect of toosendanin(TSN)on invasion and migration abilities of human ovarian cancer cells and the related mechanism.METHODS:The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations.The cell viabilty at 12,24,48,72 and 96 h after TSN treatment was measured by CCK-8 assay.Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells.The protein expression of nuclear factor-κB(NF-κB)p65,E-cadherin,N-cadherin,vimentin and Snail was determined by Western blot.RESULTS:TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells(P<0.05 ).Compared with control group ,the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly(P<0.05).In addition,the expression of NF-κB p65 and E-cadherin protein increased no-tably,followed with N-cadherin,vimentin and Snail protein decreased significantly(P<0.05).However,the inhibitor of NF-κB BAY11-7082 reversed the impact above.Compared with TSN group ,the migration and invasion abilities in TSN +BAY11-7082 group increased significantly(P<0.05).The protein expression of E-cadherin also decreased notably ,fol-lowed with the protein expression of N-cadherin,vimentin and Snail increased significantly(P<0.05).CONCLUSION:TSN inhibits the invasion and migration abilities of human ovarian cancer cells ,which is related to the inhibition of epitheli-al-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.
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AIM:To investigate the synergistic effect of toosendanin on regulating the cytotoxicity of γδ T cells to colorectal cancer cells. METHODS:γδ T cells amplified in vitro were identified by flow cytometry. Lactate dehydro-genase (LDH) release was detected to evaluate the cytotoxicity of γδ T cells and toosendanin to SW480 cells. The role of toosendanin in regulating the protein expression of Bcl-xL,Bcl-2 and MCL-1 was determined by Western blot. The effect of toosendanin on regulating the secretion of TNF-related apoptosis-inducing ligand(TRAIL) and Fas ligand(FasL) by γδ T cells was evaluated by ELISA. The mitochondrial membrane potential and apoptosis in SW480 cells treated with γδ T cells and toosendanin were analyzed by flow cytometry. The activation of caspase-9 and caspase-3 were determined by Western blot. RESULTS:CD3 and γδ T-cell receptor(TCR) were highly expressed in the γδ T cells amplified in vitro. Combina-tion with toosendanin significantly enhanced the cytotoxicity of γδ T cells to SW480 cells. Toosendanin did not influence the secretion of TRAIL and FasL secreted by γδ T cells. Toosendanin did not regulate the expression of Bcl-xL and Bcl-2 but suppressed the expression of MCL-1 in SW480 cells. In addition, enforced expression of MCL-1 obviously suppressed the synergistic effect of toosendanin on γδ T cell-induced cell death in SW480 cells. Meanwhile,co-treatment with toosendanin was able to enhance the γδ T cell-induced apoptosis and decrease of mitochondrial membrane potential. γδ T cell-depend-ent activation of caspase-9 and caspase-3 was significantly enhanced by toosendanin co-treatment in SW480 cells. CON-CLUSION:Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing the expression of MCL-1.
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Fragments of the human indoleamine 2,3-dioxygenase 1 (IDO1) gene 5'-UTR (untranslated 1 245 bp region) promoters were amplified by PCR and cloned into pGL4.20 vector in the construction of reporter vector pGL4-IDO1-luc. A549 cells were transfected with the constructed plasmid and IDO1 inhibitor screening model was established with dual-luciferase reporter assay. Based on the model, we screened natural small molecules which could down-regulate the expression of IDO1 on tumor cells. The anti-tumor activities were examined by MTT, Western blotting and lactic dehydrogenase (LDH) release assays. Toosendanin (NS-180) down regulated the IDO1 expression and inhibited IFN-γ-induced STAT1 and STAT3 phosphorylation in A549 cells. Moreover, NS-180 significantly increased the cytotoxicity of co-cultured NK cells on A549 cells in LDH release assays. In summary, NS-180 is a novel and potent IDO1 inhibitor, which has an antitumor activity for cancer immunotherapies.
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Objective: To analyze and identify the chemical constituents from the water extract of Toosendan Fructus by UPLC-ESI- Q-TOF-MS. The analysis was performed on an ACQUITY HSS T3 reverse phase column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of acetonitrile and 0.1% formic acid was used for gradient elution, the flow rate was 0.4 mL/min. Methods: Quadrupole-time of flight-mass spectrometry was applied for the qualitative analysis under positive and negative ion modes and ESI ion source was used for mass spectra. Results: The results indicated that fifteen compounds from the water extract of Toosendan Fructus had been identified by direct comparison in both positive and negative ion mass data, the element compositions analysis, and the data of the literature. They are vanillic acid, lilac acid, p-hydroxybenzoic acid, rutin, meliatoosenin E, toosendanin, Δ5,6-isotoosendanin, isotoosendanin, meliatoosenin N, meliatoosenin P, 1-deacetylnimbolinin B, meliatoosenin R, 1-O-tigloyl-1-O-debenzoylohchinal, meliatoosenin R, and nimbolinin B. Conclusion: The efficient separation ability of UPLC and high sensitive detection of MS are used in this study, which will provide the evidences for evaluating the quality of Toosendan Fructus herbs, stabilizing the curative effect in clinic, and explaining the mechanism.
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ObjectiveTo observe the effect of toosendanin on proliferation and apoptosis of human hepatoma cell line HepG2 cells,and to study the mechanism.MethodsHuman hepatocellular carcinoma HepG2 cells were randomly divided into control group(group A) does not add any medication,the observation group(group B) joined TSN 80mmol/ml,cultured cells for 24,48,72 hours.HepG2 cells proliferation inhibition rate was measured by MTT; Hepatocellular carcinoma HepG2 cell cycle and apoptosis rate was deteded by flow cytometry.ResultsTECAN determination revealed two groups of hepatocellular carcinoma cell proliferation had statictical significance ( x2 =5.33,P ＜0.05 ) ;flow cytometry revealed two groups S,G0/G1,M/G2 phase of the cell rate ( t =6.31,6.26,6.56,all P ＜0.05 ),apoptosis rate had significant difference ( x2 =6.15,P ＜ 0.05 ).ConclusionToosendanin could obviously inhibit the proliferation of HepG2 cells,and induce apoptosis of tumor cells.
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Usage of the fruit and bark of a Melia-family plant as a digestive tract-parasiticide and agricultural insecticide was recorded about two thousant years ago in ancient China. Toosendanin (TSN), a triterpenoid, is an effectual ingredient extracted from the plant. Studies have demonstrated that TSN selectively affects neurotransmitter release, effectively antagonizes botulism, induces cell differentiation and apoptosis and inhibits proliferation of various human cancer cells, inhibits feeding and dovelopment in insects and modifies K+- and Ca2+-channel activity. The research data to demonstrate that TSN inhibits K+-channel and facilitates L-type Ca2+-channel are summarized, and the mechanism of action of TSN is discussed.
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Objective:To establish an improved transonic extraction-high performance liquid chromatography(HPLC)method for separating and detecting toosendanin(TSN)in Melia toosendanin extractive.Methods:Transonic alcohol-chloroform extraction method was used to extract TSN from the bark of Melia toosendanin sieb.et zuccc.The content determination of TSN was performed on an Agilent XDB C18 Column(4.6?250 mm,5?m).Elution was performed using 45%acetonitrile-water solution as the mobile phase at a1flow rate of 1ml/min.Detection was performed at 215 nm wavelength.Results:The linear regression equation for peak area-TSN concentration(4.64～88.2?g/ml)was:A=7.616 2C-2.567 7,r=0.999 6;mean recovery of standard substance was 100.83%;RSD of precision test was 1.42%;RSD of stability test was 2.26%.Conclusion:The method is simple,rapid,stable and roducible,which can be used as a method for preparation and identification TSN of Melia toosendanin extractive in laboratory.