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OBJECTIVE：To desig n and sy nthesize poly （γ-glutamic acid ）-ampelopsin（γ-PGA-AMP），and to characterize it and evaluate its anti-tumor activity in vitro . METHODS ：Synthetic product was produced through an esterification reaction between γ-PGA and ampelopsin. The structure of synthetic product was characterized by the UV spectrophotometry ，Fourier transform infrared（FT-IR）spectroscopy，1H-NMR spectra and the quantitative elemental analysis. The content of ampelopsin in synthetic product was determined by UV absorption spectrometry at 292 nm. Using 5-FU as positive control ，MTT assay was used to determine inhibitory effects of γ-PGA-AMP and ampelopsin on human breast cancer cell MCF- 7，human liver cancer cell HepG 2 and human lung cancer cell A 549. The IC 50 was calculated. RESULTS ：The results showed that the free 7-hydroxyl group of ampelopsin and the a-carboxyl group of γ-polyglutamic acid had been esterified to obtain γ-PGA-AMP；the yield of γ-PGA-AMP was 55.7％，and the content of ampelopsin was 32.3％. The inhibitory effect of γ-PGA-AMP and ampelopsin on MCF- 7，HepG2 and A 549 cells was obvious. IC 50 of γ-PGA-AMP（to 3 above tumor cells ）were 40.19，28.29 and 55.23 μg/mL，those of ampelopsin were 105.30，81.23，130.10 μg/mL，those of 5-FU were 24.72，87.98，30.99 μg/mL，respectively. CONCLUSIONS ：γ-PGA-AMP with anti-tumor effect in vitro is synthesized successfully ，and its anti-tumor effect is stronger than that of ampelopsin.
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Objective: To investigate the inhibitory effects of ampelopsin (AMP) on proliferation, autophagy and apoptosis to human colon cancer cells and its possible mechanism. To observe the function of autophagy in the apoptosis of colon cancer cells as well. Methods: The lentivirus infection method was adopted here to construct double fluorescence [green fluorescent protein (GFP) and red fluorescent protein (RFP)] labeled microtubule-associated protein light chain 3 (LC3) overexpressed recombinant HCT116-RFP-GFP-LC3 cells. The GFP and RFP labeled LC3 expression in HCT116-RFP-GFP-LC3 cells were observed by fluorescence microscope, and Western blotting was used to detect the expression of LC3 in HCT116-RFP-GFP-LC3 cells at the same time. The proliferation inhibition of recombination HCT116-RFP-GFP-LC3 cells and parental HCT116 cells treated with different concentrations of AMP (0, 50, 100, 175, 250, 350, 400 and 450 μg/mL) was detected by CCK-8 assay and the cell morphology was observed. The apoptotic rate of HCT116 cells treated with AMP (0, 25 50, and 75 μg/mL) was detected by FCM method, and the apoptotic-related proteins Bax, nuclear factor-kappa B (NF-κB) and Bcl-2 expression levels were detected by Western blotting. Autophagy induction was detected by counting fluorescence dots triggered by transformation of LC3to LC3Ⅱ visualized by laser confocal microscopy after HCT116-RFP-GFP-LC3 cells treated with AMP (0, 25 50, and 75 μg/mL) alone or combined with bafilomycin A1(Baf A1) (an autophagy inhibitor) (20 nmol/L). After the HCT116 cells were treated with AMP (25 μg/mL) alone or combined with Baf A1 (20 nmol/L), the apoptotic rate was detected by FCM method, and the expression level of autophagy-related proteins LC3Ⅱ and p62 as well as apoptotic-related proteins were detected by Western blotting. Results: RFP-GFP-LC3 could stably express in HCT116-RFP-GFP-LC3 cells, indicating that HCT116-RFP-GFP-LC3 recombinant cells were successfully constructed. The results of CCK-8 assay suggested that AMP could significantly inhibit the proliferation of recombinant and parental cells in a concentration-dependent manner. Meanwhile, there was no significant difference in cell morphology or cell proliferation inhibition rate between these two cells (P > 0.05). The apoptosis rate of HCT116 cells increased remarkably along with the enhancement of AMP concentration which means there is a concentration-dependent manner (all P < 0.05). The expression levels of pro-apoptotic protein Bax and NF-κB were increased, and the apoptosis inhibitory protein Bcl-2 was down-regulated (all P < 0.01). The confocal microscopy results showed that with the increase of AMP concentration, the numbers of autophagy points were significantly increased (P < 0.01), and the expression level of autophagy protein LC3Ⅱ was up-regulated, indicating that AMP could trigger autophagy on colon cancer cells. Comparing with AMP alone group, AMP combined with Baf A1 could increase the apoptosis rate of HCT116 significantly (P < 0.05), and the expression level of apoptotic protein Bax promoted remarkably (P < 0.05). The expression level of the apoptosis inhibitory protein Bcl-2 demoted as well. All these results suggest that autophagy blocking could promote AMP induced apoptosis. Conclusion: AMP can inhibit the proliferation of colon cancer cells HCT116, inducing autophagy and apoptosis; inhibition of autophagy can promote AMP-induced apoptosis on colon cancer cells.
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OBJECTIVE： To study the antimicrobial activity of ampelopsin combined with 8 kinds of antibiotic against Pseudomonas aeruginosa （PA） in vitro. METHODS： Chessboard trace dilution method was used to determine minimum inhibitory concentration （MIC） of ampelopsin combined with ceftriaxone， cefoperazone/sulbactam， piperacillin/tazobactam， cefoperazone， ciprofloxacin， amikacin， piperacillin and cefepime to PA strain ATCC27853 and 7 isolated strains PA135， PA216， PA276， PA281， PA291， PA314 and PA319. Fractional inhibitory concentration index （FICI） was used to evaluate its effects of drug combination. Clinically isolated strain PA319 were taken as target strain and then divided into normal control group， ampelopsin alone group， antibiotics alone group and ampelopsin+antibiotic combination group. Using MIC of ampelopsin and antibiotics during drug combination as active concentration， the number of colonies cultured for 0， 4， 8， 12 and 24 h was counted， and the time-sterilization curve was drawn. RESULTS： For above 8 kinds of strains， MIC of ampelopsin alone was 128-256 mg/L； FICI of ampelopsin combined with ceftriaxone， cefoperazone/sulbactam， piperacillin/tazobactam， cefoperazone， ciprofloxacin， amikacin， piperacillin and cefepime to 8， 8， 7， 6， 4， 4， 6 and 6 strains were equal to or lower than 1， respectively. In time-antibacterial curve， compared with antibiotics alone， the number of colonies decreased by 2.65， 2.30， 0.42， 0.47， 0.53， 1.19， 1.74， 1.04 lgCFU/mL respectively after ampelopsin combined with ceftriaxone， cefperazone/sulbactam， piperacillin/tazobactam， cefoperazone， ciprofloxacin， amikacin， piperacillin and cefepime. CONCLUSIONS： Ampelopsin combined with ceftriaxone and cefoperazone/sulbactam show better antibacterial effect on PA.
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This research is aimed to investigate the effect of ampelopsin on apoptosis and migration of human hepatoma SMMC-7721 cells and explore the molecular mechanism. SMMC-7721 cells were pretreated with different doses of ampelopsin and cells proliferation was detected by CCK8 kit. Cell morphology was observed under an inverted microscope. Nuclear morphology was detected by DAPI staining. Apoptotic rate was detected by Annexin V-FITC/PI flow cytometry. Migration and invasion were detected by Transwell and scratch healing test. Western blotting was used to detect cleavage of poly ADP-ribose polymerase (PARP), expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), E-cadherin, and N-cadherin, and phosphorylation of ERK, P38 and JNK in MAPKs pathway. Our results showed that ampelopsin significantly inhibited proliferation and induced apoptosis of SMMC-7721 cells, with half inhibition dose (IC50) for 24 h was 38.98 μg·mL-1. With 50 μg·mL-1 ampelopsin treatment, typical apoptotic morphological changes occurred, such as cell detachment, shrinkage and nuclear condensation. Apoptotic rate increased from 15% to 55.1%, with PARP cleavage significantly increased. In addition, treatment of ampelopsin reduced scratch healing of cells and transmembrane cells number. The expression levels of MMP-2 and MMP-9 were decreased. Further analysis of EMT-related proteins showed that after ampelopsin treatment, E-cadherin was up-regulated and N-cadherin was down-regulated. During ampelopsin treatment, ERK reached its peak of activation after 1 h, while the maximum activation time of JNK was 12 h. Meanwhile, P38 was activated within 4 h, with the highest point at 2 h. But after 4 h, ampelopsin inhibited phosphorylation of P38. These results indicated that ampelopsin induced apoptosis and reduced migration through activating MAPKs pathway and reversing EMT process in SMMC-7721 cells. This work provides a mechanistic basis for utilizing ampelopsin for anti-hepatocarcinoma treatment.
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<p><b>OBJECTIVE</b>To evaluate the cytotoxic effects of ampelopsin sodium (Amp-Na) and carboplatin (CBP) used alone or in combination on human non-small cell lung cancer (NSCLC) cells SPC-A1 in vitro and its related mechanism.</p><p><b>METHODS</b>Cytotoxic effects were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The synergistic effects of the drugs were calculated with coefficient of drug interaction (CDI). Cell cycle was determined by flow cytometry (FCM). The levels of p53, p21, cyclinE, cyclinD1, and phosphorylated cyclin-dependent kinase-2 (p-CDK2) were evaluated by Western blot.</p><p><b>RESULTS</b>Amp-Na (6.25-200 μg/mL) and CBP (3.13-100 μg/mL) alone exhibited prominent cytotoxic activity in a concentration-dependent manner on SPC-A1 cells with 50% inhibitive concentration values of 57.07±14.46 and 34.97±6.30 μg/mL, respectively. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone (P<0.05 or 0.01). The CDI analysis confirmed the synergy of Amp-Na and CBP on inhibiting cancer cell viability across a wide concentration range (CDI <1). FCM and Western blot showed that synergistic cytotoxic effects of Amp-Na and CBP were related to Garrested which mainlym ediated by p 21 through the inhibition of CDK2 activity independent of the p53 tumor suppressor pathway.</p><p><b>CONCLUSIONS</b>Amp-Na exhibits anticancer activities and enhances the antitumor activities of CBP through up-regulation of p21 and inhibition of CDK2 activity in human NSCLC cells SPC-A1. These results suggest that Amp-Na may be applied to enhance the anticancer action of CBP.</p>
Subject(s)
Humans , Antineoplastic Combined Chemotherapy Protocols , Pharmacology , Carboplatin , Pharmacology , Carcinoma, Non-Small-Cell Lung , Drug Therapy , Pathology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Drug Synergism , Flavonoids , Pharmacology , Lung Neoplasms , Drug Therapy , PathologyABSTRACT
To improve the solubility and antitumor activity of ampelopsin, ampelopsin-loaded nanomicelles from the mixture of pluronic F127 and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS1000) were prepared by film-thin hydration method, in order to optimize the process conditions and physicochemical properties. The antitumor activities against MCF-7 cells between ampelopsin and nanomicelles were compared by MTT method, respectively. The results showed that the optimal nanomicelles were round with the nanometric size of (22.6±0.5) nm, encapsulation efficiency rate of (80.42±1.13)%, and drug-loading rate of (4.41±0.26)%. The solubility of ampelopsin in mixed nanomicelles significantly increased by 16 times. In different release media, the mixed nanomicelles could release more than 90% of drug in 8 h, and showed stronger cytotoxicity and inhibition against MCF-7 cells (P<0.01). The mixed nanomicelles can be used as new drug delivery system of ampelopsin.
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OBJECTIVE:To study the inhibitory effects of ampelopsin sodium (AMP-Na) combined with carboplatin on the proliferation of Lewis cell of lung cancer. METHODS:6.25,12.5,25,50 and 100 μg/ml AMP-Na and 3.125,6.25,12.5,25 and 50 μg/ml carboplatin were used to culture the cells for 4 h,and the cell viability was determined and the inhibition rate and half in-hibition concentration(IC50)were calculated. 12.5,25,50 and 100 μg/ml AMP-Na and 12.5 μg/ml carboplatin were used to culture the cells for 12 h,and the flow cytometry was used to determine the expression of Caspase-3. RESULTS:AMP-Na with serial con-centrations combined with carboplatin with serial concentrations had obvious inhibitory effects on the cell proliferation. With the in-crease of mass concentration,the IC50 of carboplatin on the Lewis cells was gradually decreased;when the AMP-Na of 6.25-50μg/ml was combined with carboplatin of 3.125-25 μg/ml,it showed the strongest inhibitory effects on the Lewis cell proliferation. When cells were cultured with AMP-Na and carboplatin for 24 h,the expression of Caspase-3 increased significantly. CONCLU-SIONS:AMP-Na combined with carboplatin has synergistic inhibitory effect on the Lewis cell proliferation by a mechanism that may be related to the apoptosis induced by Caspase-3 activated by AMP-Na.
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Objective To investigate the effect of ampelopsin on apoptosis induction and cell growth inhibition in Human Colon cancer SW480 cells in vitro.Methods Treated with ampelopsin at several concentrations, MTT and flow cytometry was used to detect the inhibition rate and apoptotic rate of SW480 cells.Western-blot was used to investigate expression of Bcl-2 family pro-tein.Results Significant difference of cell growth inhibition rate was observed among all groups after treated with ampelopsin ( p0.05).The similar results were observed in the experiment on apoptosis induction.Level of Bcl-xL was significantly up-regulated.Level of Bax, Bid, Caspase-3, p-Ca-pase-9 and Caspase-9 was significantly down-regulated after treatment of ampelopsin.Conclusion Ampelopsin can inhibit cell growth and induce apoptosis of SW480.Bcl-2 family protein might be involved in the progress.
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Aim To investigate the cytotoxic effect and mechanism of ampelopsin sodium ( AMP-Na ) on hu-man lung adenocarcinoma cell line GLC-82 alone or combined with carboplatin ( CBP ) . Methods The cytotoxic effect of human lung adenocarcinoma cell line GLC-82 was investigated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide ( MTT ) colori-metric assay. Ultrastructure change of apoptotic GLC-82 cells was observed with transmission electron micro-scope. The changes of the cell apoptosis and the ex-pression of caspase-3 were analyzed with flow cytome-ter. Results Combined with AMP-Na, the IC50 of CBP decreased from (17. 10 ± 4. 78) mg·L-1 to tron microscope and flow cytometric analysis, the apop-tosis and necrosis ratios also increased in the combina-tion group. The necrosis ratios increased from (2. 56 ± 0. 41 )% to ( 71. 83 ± 5. 43 )% ( P<0. 01 ) . The ex-pression of caspase-3 was increased significantly after treated with AMP-Na or combined with CBP. Conclu-sions There is a synergistic cytotoxic effect on GLC-82 cells treated with AMP-Na combined with CBP. Ap-optotic cells and necrotic cells are found in GLC-82 cells treated with AMP-Na alone or combined with CBP. One of the mechanisms to induce apoptosis is probably that activation of caspase-3 mediates signal transduction pathway in cells.
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Objective: To study the chemical constituents in Vitis thunbergii var. taiwaniana specially grown in Taiwan, China. Methods: The compounds were isolated by repeated HP20 macroporous adsorption resin column combined with Sephadex LH-20, ODS, and silica gel column chromatography. Their structures were identified on the basis of extensive spectroscopic data analysis and by comparison of their spectral data reported. Results: Twelve compounds were identified as resveratrol (1), trans-ε-viniferin (2), (7R, 8R)-threo-4, 7, 9, 9'-tetrahydroxy-3, 3'-dimethoxy-8-O-4'-neolignan 7-O-β-D-glucopyranoside (3), (7S, 8R)-urolignoside (4), schizandriside (5), vitisin A (6), vitisin B (7), davidiol A (8), 3, 5, 4'-trihydroxystilbene 4'-O-β-D-glucopyranoside (9), ampelopsin C (10), (7R, 8S)-dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucopyranoside (11), and epicatechin (12). Conclusion: Compounds 3-5 are separated from the plants of Vitis L. for the first time, and compounds 8, 9, 11, and 12 are separated from V. thunbergii var. taiwaniana for the first time.
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Aim To investigate the effects of ampelopsin on induction of apoptosis in human hepatocellular carcinoma Bel-7402 cells.Methods Bel-7402 cells were treated with ampelopsin with different concentrations for 24,48 and 72 h.The cell proliferation was detected by MTT assay.The morphological change of cells was observed through microscope observation by fluorescence staining.DNA fragmentation was visualized by agarose gel electrophoresis.The apoptosis rate was analyzed by flow cytometry.The expressions of caspase-3,Bcl-2 and Bax protein were detected by Western blot.Results Ampelopsin inhibited the proliferation of Bel-7402 cell line in a dose-and time-dependent manner.The IC50 values were 89.6?16.1,36.2?6.5 and 15.3?3.0 mg?L-1 at 24,48 and 72 h,respectively.The fluorescence microscope showed clearly cell apoptosis with apoptotic body.Agarose gel electrophoresis result showed that Bel-7402 treated with ampelopsin produced a DNA ladder band.The sub-G1 peak was detected and resulted in dose-and time-dependent increasing of the population of sub-G1 DNA content by flow cytometry.The expression of Bcl-2 protein was down-regulated,while the expression of Bax protein was up-regulated.The pro-caspase-3 protein was down-expressed and activated.Conclusions Ampelopsin could inhibit Bel-7402 proliferation through inducing cell apoptosis.The mechanism might be that ampelopsin could directly or indirectly enhance the level of anti-apoptosis protein Bcl-2 and decrease the level of apoptosis protein Bax.The pathway of pro-caspase-3 activated was initiated and effector caspase-3 was sequentially activated.