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ObjectiveThe aim of this study is to investigate the role of salidroside in regulating the miR-1343-3p/MAP3K6 （mitogen-activated protein kinase kinase kinase 6）/MMP24 （membrane-type matrix metalloproteinase 24） signaling pathway to inhibit gastric cancer cell proliferation and migration. MethodsHuman gastric cancer cells （MGC-803） were divided into several groups based on different salidroside concentrations： a control group （0 μmol/mL）， a low-dose group （6 μmol/mL）， a medium-dose group （12 μmol/mL）， and a high-dose group （24 μmol/mL）. The anti proliferative effects of salidroside on human gastric cancer cells were evaluated by CCK-8 assay. Clonogenic assay was used to examine the effects of salidroside drugs on the clonogenic ability of human gastric cancer cells. Transwell assay was performed to detect the effect of salidroside on the invasive ability of human gastric cancer cells. Cell scratch assay was performed to detect the effect of salidroside on the migration ability of human gastric cancer cells. The miRNA expression profile was analyzed by using RNA-seq in cancer cells for 24 h after salidroside treatment. The differentially expressed miRNAs were clustered and their target genes were predicted. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） were used to analyze and predict the functions of these target genes， and the interaction networks were established. Immunocytofluorescence was used to detect the expression of target proteins， and the transcription of candidate genes was detected by q-PCR. ResultsCCK-8 cytotoxicity experiments showed that salidroside inhibited the proliferation of MGC-803 cells （P < 0.01）. Cell cloning experiments showed that salidroside reduced the clonal formation capacity of MGC-803 cells （P < 0.000 1）. Cell invasion experiments showed that salidroside reduced the MGC-803 cell invasion capacity （P < 0.000 1）. Cell scratch experiments showed that salidroside reduced the cell migration capacity （P < 0.000 1）. RNA-seq findings showed that the expression of 44 miRNAs changed significantly after salidroside treatment in cancer cells （P < 0.05）. Bioinformatic analysis showed that there were 1 384 target mRNAs corresponding to the differentially expressed miRNAs， and the expression of the tumor suppressor miR-1343-3p was significantly upregulated after salidroside treatment （P < 0.01），and resulted in down-regulated transcription of MAP3K6 and MMP24 genes which are related to the proliferation and migration of cancer cells （P < 0.05）. Immunofluorescence experiments demonstrated that salidroside reduced protein expression levels in MAP3K6 and MMP24 genes （P < 0.000 1）. q-PCR experiments showed that salidroside reduced the mRNA expression level of MAP3K6 and MMP24 genes （P < 0.000 1）， while miRNA expression in miR-1343-3p gene was upregulated （P < 0.000 1）. ConclusionSalidroside regulates the miRNA-1343-3p/MAP3K6/MMP24 signaling molecules to inhibit proliferation and invasion of gastric cancer cells.
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Objective: The aim of this study is to probe in the inhibitory effects of ginsenoside Rg3 on the expression of hypoxia-induced factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in human gastric cancer cells. Materials and Methods: Human gastric cancer BGC823 cells were divided into the control group and experiment group, and expression levels of HIF-1α and VEGF were detected by immunocytochemistry and Western blot after cells were cultured under hypoxia for different durations. Results: Under hypoxia, expression of HIF-1α and VEGF in human gastric cancer BGC823 cells showed an increasing trend, and that was remarkably lower in experiment group than in the control group after applying Rg3, which was obvious at 12 and 24 h (P < 0.05). Conclusion: Rg3 can inhibit expression of HIF-1α and VEGF in human gastric cancer cells and may influence abdominal implantation metastasis of gastric cancer through inhibiting its expression
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OBJECTIVE： To study the effects of ivermectin on the migration and invasion of human gastric cancer cell lines BGC-823 and MGC-803 and its mechanism. METHODS： After treated with 0， 2.5， 5， 10， 20， 40 μmol/L ivermectin for 24 h， inhibitory rate of human gastric cancer cell lines BGC-823 and MGC-803 were detected by MTT assay. Effects of 5 μmol/L ivermectin and phosphate buffercontaining 0.67‰ dimethyl sulfoxide （control group） for 24 h on the migration and invasion of` gastric cancer cells BGC-823 and MGC-803 were observed by Transwell chamber invasion assay.Western blot assay was used to detect the protein expression of TGF-β1， TGF-βR， Smad2 and Smad3 in epithelial-mesenchymal transition （EMT） markers E-cadherin， N-cadherin， Vimentin， Snail and EMT transduction pathway TGF-β/smad of BGC-823 and MGC-803 cells after treated with 5， 10 μmol/L ivermectin and phosphate buffercontaining 0.67‰ dimethyl sulfoxide （control group） for 24 h. RESULTS： Ivermectin could inhibit the growth of BGC-823 and MGC-803， inhibitory rate of it was positively correlated with its concentration. Compared with control group， the number of migration and invasion BGC-823 and MGC-803 cells were decreased significantly after treated with 5 μmol/L ivermectin （P＜0.01 or P＜0.001）； the expression of E-cadherin protein was enhanced significantly in BGC-823 and MGC-803 cells after treated with 5 and 10 μmol/L ivermectin （P＜0.05 or P＜0.01 or P＜0.001）； the protein expression of N-cadherin， Vimentin， Snail， TGF-βR， Smad2 and Smad3 were decreased significantly （P＜0.05， P＜0.01 or P＜0.001）； protein expression of TGF-β1 was decreased significantly after treated with 10 μmol/L ivermectin （P＜0.05）. CONCLUSIONS： Ivermectin can significantly inhibit the migration and invasion of gastric cancer cells BGC-823 and MGC-803， and inhibiting the biological activity of EMT by reducing the expression of TGF-β/smad pathway is one of the mechanisms that inhibit the migration and invasion of gastric cancer cells.
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Objective: To observe the effect of Weichang'an on the tumor growth and lymph node metastasis, and RUN and FYVE domain-containing protein 3(RUFY3),Zinc finger protein 1(SNAI1),vascular endothelial growth factor(VEGF),epithelial-mesenchymal transition(EMT)-related proteins in nude mice with subcutaneous xenograft tumor human gastric carcinoma MKN45, so as to discuss the mechanism of Weichang'an on MKN45 human gastric metastasis. Method: The nude mice model of human gastric carcinoma MKN45 cells was established and randomly divided into normal saline group (0.5 mL/mouse), Weichang'an granule group (3.54 g·kg-1) and Weichang'an decoction group (35.49 g·kg-1). The tumor weight and volume of axillary lymph nodes in each group were observed. The morphology of lymph nodes in each group was detected by hematoxylin-eosin (HE) staining. The expressions of related proteins were detected by immunohistochemistry, including RUFY3,SNAI1,VEGF,E-cadherin,N-cadherin,Vimentin. Result: Compared with the normal saline group, the tumor weight and volume of axillary lymph node were decreased (PPPConclusion: Both Weichang'an granule and Weichang'an decoction can inhibit the tumor growth and metastasis of axillary lymph nodes, obviously down-regulate the expressions of RUFY3,SNAI1,VEGF,N-cadherin,Vimentin and up-regulate the expression of E-cadherin in human gastric MKN45 subcutaneous transplanted tumor in nude mice. This suggested that Weichang'an may inhibit the tumor metastasis through regulation expressions of RUFY3,SNAI1,VEGF and EMT-related proteins.
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Objective:To define the anti-gastric cancer activity in vitro of petroleum ether fraction of Boehmeria nivea root and reveal the material basis of its efficacy, so as to lay the foundation for the development and utilization of B. nivea root. Method:Methyl thiazolyl tetraolium(MTT) method was used to evaluate the inhibitory rate and time-dose relationship of petroleum ether fraction of B. nivea root with different doses and delivery times on human gastric cancer HGC-27 cells. Flow cytometry was used to detect the change of cell apoptosis and cell cycle after petroleum ether fraction of B. nivea root acted on human gastric cancer HGC-27 cells. GC-MS was used to detect the components of petroleum ether fraction of B. nivea root. Result:Experiment data showed significant cell proliferation inhibition in an obvious time-dose-effect manner, with statistically significant differences (PB. nivea root. The effect of petroleum ether fraction of B. nivea root on human gastric cancer HGC-27 cells could induce apoptosis,which affects the normal changes of cell cycle. The percentage of cells was decreased significantly in G0/G1 phase,and that in S phase was significantly increased. GC-MS was used to identify 26 chemical constituents in petroleum ether of B. nivea root,including sitosterol and stigmasterol. Conclusion:Petroleum ether fraction of B. nivea root is the active anti-gastric cancer part,and its main effective component is sterol compounds. This lays the foundation for the rational application of B. nivea root in clinic and the further research in tis anti-tumor effect.
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@# Objective: To observe the effect of ursolic acid (UA) on autophagy and apoptosis of gastric cancer cell line MGC-803, and to explore the mechanism of UA-induced autophagy of MGC-803 cells based on PI3K/AKT/mTOR signaling pathway. Methods: Human gastric cancer cell line MGC-803 was cultured in vitro and divided into blank control group, UAintervention group and UA+3-MA group. The cell apoptosis in each group was detected by flow cytometry. Cell autophagy was detected by double fluorescence mRFPeGFP-LC3 plasmid transfection method. The mRNA expression levels of LC3B, BAX and Bcl-2 were detected by qPCR. The protein expression levels of PI3K type I, p-AKT, p-mTOR, ULK1, LC3B, BAX and Bcl-2 were detected by WB. Results: Flow cytometry showed that the cell apoptotic rate of UA intervention group was significantly higher than that of blank control group (P<0.05). Compared with UAintervention group, the apoptotic rate in UA+ 3-MAgroup was significantly reduced (P<0.05). The double fluorescence mRFP-eGFP-LC3 plasmid transfection method showed that the green and red fluorescent bright spots in UA intervention group increased significantly compared with the blank control group (P<0.05), and the green and red fluorescent bright spots in UA+3-MA group were significantly reduced compared with UA intervention group (P<0.05). Real-time quantitative PCR and WB method showed that compared with the blank control group, the mRNAand protein expressions of BAX and LC3B, and ULK1 protein were significantly increased in UA intervention group, while the mRNA and protein expressions of Bcl-2, and the protein expressions of PI3K, p-AKT and p-mTOR were significantly decreased in UA intervention group (all P<0.05); Compared with UA intervention group, mRNA and protein expressions of BAX and LC3B were significantly down-regulated and the mRNAand protein expressions of Bcl-2 were significantly up-regulated in UA+3-MA group (all P<0.05), while protein levels of PI3K, p-AKT, p-mTOR and ULK1 were not significantly changed in UA+3-MA group (P>0.05). Conclusion: UA can promote apoptosis of MGC-803 cells via inducing autophagy, which may be related to UA's involvement in regulating the expressions of PI3K/AKT/mTOR signaling pathway-related proteins.
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OBJECTIVE: To screen the best combination of extractum of Robinia-living trametes and chemotherapy and investigate the action mechanism of Robinia-living trametes against the apoptosis of human gastric cancer cell MGC803. METHODS: MGC803 Cells were treated with different concentrations of Robinia-living trametes and chemotherapy drugs (5-Fu and paclitaxel) in vitro. The inhibitory rate of cells was measured by MTT assay. Morphological changes were observed with inverted microscope. The apoptosis rate of MGC803 cells which were treated with combination of Robinia-living trametes (0.2 mg·mL-1) and 5-Fu (2.5 μg·mL-1) was detected by FCM. The protein expression of P53 and p-Akt in MGC803 cells which were treated with combination of Robinia-living trametes (0.2 mg·mL-1) and 5-Fu (2.5 μg·mL-1) was detected by Western blot. RESULTS: The viability of MGC803 cells was reduced by Robinia-living trametes and chemotherapy drugs (5-Fu and paclitaxel) in a concentration- and time-dependent manner (P<0.01). Under reverse microscopy, cell body shrinking, nuclear pyrosis, and nuclear fragmentation were observed. The higher concentration, the longer treatment time, the more cells died. Compared with monotherapy, the combination of Robinia-living trametes and chemotherapy could reduce the survival rate of MGC803 cells. The protein expressions of P53 in MGC803 cells treated with combination of drugs was up-regulated, while that of P-Akt was down-regulated. CONCLUSION: The apoptosis of MGC803 cells in vitro may be induced by the inhibitory effect of the combination of Robinia-living trametes and 5-Fu on PI3K/Akt signaling pathway. Combination therapy of Robinia-living trametes and 5-Fu is potentially more effective in inhibition of tumor cells than monotherapy of Robinia-living trametes.
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ABSTRACT:Objective To investigate the effects of ω-3 polyunsaturated fatty acids on the proliferation of human gastric cancer cell line AGS and the possible mechanisms.Methods Human gastric cancer line AGS and human microvascular epithelial cell HMEC-1 were treated with different concentrations of docosdhexaenoic acid (DHA)and eicosapentaenoic acid (EPA).The inhibition of cell proliferation was evaluated by MTT assay and cell morphology.Flow cytometry was used to detect the cell cycle change.The expressions of mitochondrial respiratory membrane protein complex Ⅰ,Ⅱ and V were analyzed with Western blot.Results DHA and EPA could markedly inhibit the proliferation of AGS in significant time-dependent and concentration-dependent manners (P 0.05).Conclusion ω-3 PUFAs can selectively inhibit the growth and proliferation of human gastric cancer cell line AGS.These effects may be as-sociated with arresting cell cycle in G0/G1 phase and inhibiting the energy metabolism of AGS cells.
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OBJECTIVE: To investigate the effect of galangin on the anti-proliferation and apoptosis of gastric cancer SGC-7901 cells, and possible mechanisms. METHODS: Proliferative activity of SGC-7901 cells was measured by MTT assay while that cell cycle progression and apoptosis of galangin-treated SGC-7901 cells were analyzed by flow cytometry, morphologic observation and mitochondrial membrane potential (MMP) analysis. RESULTSS The evaluated results showed that when galangin was added into cell culture in 40-200 μmol · L-1, proliferative inhibition occurred and was observed as a dose- and time-dependent manner. The calculated IC50 values for galangin were 160, 100 and 70 μmol · L-1 when the treatment time was 24, 48 and 72 h, respectively. Flow cytometry analysis revealed that the proportion of the cells in the G2/M phase was significantly enhanced from 4.40(control group) to 18.31 (treatment group) by a treatment time of 24 h. CONCLUSION: The cells in treatment group showed typical apoptotic morphology and a decrease in MMP. At the same time, the percentage of the apoptotic cells significantly increased from 2.6% or 4.3% (control group) to 27.4% or 65.6% (treatment group), when 160 μmol · L-1 galangin treated SGC-7901 cells for 24 or 48 h. These mentioned results indicate that galangin can inhibit the proliferation and induce apoptosis of the SGC-7901 cells by perturbation in cell cycle progression and mitochondrial dysfunction.
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This study was aimed to investigate effect of aconite alkaloids on proliferation and apoptosis of hu-man gastric cancer cell line SGC-7901 . Effects of different concentrations of aconite alkaloids on proliferation of human gastric cancer cell line SGC-7901 were investigated with MTT assay; induced apoptosis and cell cy-cle blocking were detected with flow cytometer ( FCM ) . The results showed that the IC50 of aconite alkaloids on human gastric cancer cell line SGC-7901 was 0 . 2318 ± 0 . 0022 , 0 . 1601 ± 0 . 0227 , 0 . 1031 ± 0 . 0231 mg/ml at 24 h , 48 h and 72 h , respectively , compared to the control group with significant difference ( P <0.01). When the aconite alkaloids concentration was 0.8 mg/ml, it appeared with obvious apoptosis. The apop-tosis rate was ( 59 . 38 ± 5 . 05 )%. The FCM detection showed that compared with control group , the percentage of S-phase cells increased in the treatment group . And a typical sub-diploid peak appeared before G0 / G1 phase . It was concluded that aconite alkaloids can inhibit the proliferation of human gastric cancer cell line SGC-7901 in vitro, induce the apoptosis of cells and make SGC-7901 cells remain in S phase.
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Objective To detect the effect of osteopontin (OPN) siRNA on the growth,invasion and apoptosis of human gastric cancer cell line MKN28. Methods The eukaryotic expression vector of siRNA specific for OPN was constructed by liposome transfection,and the silencing effect was identified by fluorescence quantitative PCR and immunofluorescence staining. According to the inhibitory effect of the siRNA vectors,the experiment was divided into control group,non-specific OPN siRNA group,24-hour specific OPN siRNA group,36-hour specific OPN siRNA group,and 48-hour specific OPN siRNA group. The cell proliferation,apoptosis,migration and adhesion was finally detected among control group and OPN siRNA groups at different time points. Results For 48-hour specific OPN siRNA group,the expression of OPN in MKN28 cells was the lowest. Accordingly,the cell viability was decreased and the cell proliferation inhibitory rate was reduced to 30.20%; the ability of migration and adhesion was decreased,the number of cells attached on stromal cell membrane was much lesser (21.8?6.9 vs 42.0?9.4 in control group),and the ratio of cell adhesion on basement membrane matrix and fibronectin was lower [basement membrane matrix: (41.5?8.4)% vs (20.5?4.5)%; fibronectin: (25.3?4.5)% vs (14.6?2.5)% in control group],as well as the cell apoptotic rate was increased compared with the control group. Conclusion OPN gene silencing can inhibit the growth and invasion,as well as accelerate apoptosis in human gastric cancer MKN28 cells.
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We used retrovial vector LXSN to construct recombinant retroviral vector with mutant membrane bound TNF - ? gene. The vectors were introduced into packaging cell line, CRIP cells. The G418-resistant colonies were selected and the supernatants of the colonies were used to determine the virus titers. The titer of virus was 1? 104CFU/ml and the retroviral vectors were used to tranduced the human gastric cancer cell line, MGC-803 cells. The results of southern blot assay showed thai the targel gene had integrated into the genomic DNA of MGC-803T. MGC - 803Tcells were ablc to kill L929 cell line, but the parent cell line showed no cytotoxicily to the cells at all. There was no any variance in the morphological appearance and growlh rate in vilro of MGC-803N and MGC-803T cells. The results of inoculation in nude mice with these cells indicated that MGC-803T cells showed a considerable decrease in size of tumor. These results suggested that the retroviral vectors expressing mulant TNF -? gene were successfully construed. MGC - 803T cells showed cytotoxicily slrongly lo L929 cell line in vilro and lumorigenicity .
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Objective: To explore the growth inhibition and apoptosis-inducing effects of apigenin in human gastric cancer SGC-7901 cells. Method: MTT and flowcytometry were used to detect the growth inhibition and cell cycle distribution in apigenin-treated SGC-7901 cells respectively. DAPI fluorescence staining and DNA ladder assay were applied to study the pro-apoptosis effects of apigenin. The expression of apoptosis relative proteins, caspase-3 and Bcl-2, were analyzed by using Western blotting. Results: Apigenin treatment significantly inhibited the growth of human gastric cancer cell SGC-7901 and markedly caused their apoptosis following activation of caspase-3 and downregulation of Bcl-2 protein expression. Conclusion: The activation of caspase-3 and downregulation of apoptosis relative protein Bcl-2 expression were the possible mechanism of apigenin induced growth inhibition and apoptosis in SGC-7901 cells.