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Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 08 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402.
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Objective: To investigate the effects and the underlying mechanisms of evodiamine (EVO) on apoptosis of hepatocellular carcinoma (HCC) BEL-7402 cells. Methods: Cell counting kit-8 (CCK-8) assay was used to detect the effect of EVO on proliferation activity of BEL-7402 cells. Hoechst 33258 staining was used for observing morphological changes of apoptosis. The cell apoptosis and cycle distribution were analyzed by flow cytometry. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay were used to detect the expression levels of key genes from Hippo-YAP pathway in HL-7702 and BEL-7402 cells, including mammalian STE20-like protein kinase 1/2 (MST1/2), large tumor suppressor 1 (LATS1), and Yes-associated protein (YAP), then to examine the effect of EVO on the expression levels of these genes in BEL-7402 cells. The effect of EVO on the expression of YAP in hepatocellular carcinoma BEL-7402 cells was observed by immunofluorescence assay. Results: The proliferation of BEL-7402 cells were significantly inhibited by EVO in a dose- and time-dependent manners. Hoechst 33258 staining showed that EVO induced BEL-7402 cell typical apoptotic morphology, such as nuclear chromatin concentration and edge accumulation. Besides, flow cytometry tests showed that BEL-7402 cell apotosis were increased and cell cycle arrested in G2/M phase after being treated with EVO (P < 0.01). qRT-PCR and Western blotting showed that the expression level of MST2 and LATS1 were lower in BEL-7402 cell (P < 0.05), while the transcription and protein levels of YAP were significantly higher (P < 0.05). EVO could activate Hippo signal pathway, upregulate the expression of MST2 and LATS1 and then inhibit the expression of YAP in BEL-7402 cell (P < 0.05). Immunofluorescence assay also validated that EVO would significantly inhibit the overexpression of YAP in BEL-7402 cell (P < 0.01). Conclusion: EVO can induce the apoptosis of BEL-7402 cells, which may be through activating Hippo signal pathway and then down-regulate the expression of YAP.
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Objective:To investigate effects of miR-503 on cisplatin sensitivity in BEL-7402 cells by targeting of bcl-2.Methods:MiR-503 and bcl-2 mRNA expression levels in hepatocellular carcinoma cells were measured by real-time quantitative (qRT)-PCR;Bcl-protein level was detected by Western blot;miR-503 mimics were transiently transfected to the BEL-7402 cells by liposome transfection;potential target genes of miR-503 were predicted by Bioinformatics software;miR-503 potential targets were validated by dual luciferase activity;and the cell viability was measured by MTT assay.Results:MiR-503 level was down-regulated and Bcl-2 protein expression level was up-regulated in BEL-7402 cells compared with HL-7702 cells.MiR-503 could interact with bcl-2 and inhibit its expression.Cell vitality with miR-503 transfection was significantly reduced compared to that in the negative control.Conclusion:MiR-503 may enhance the sensitivity of BEL-7402 cells to cisplatin and inhibit the cell proliferation by targeting bcl-2.
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OBJECTIVE To evaluate the effect of single traditional Chinese medicine(TCM) herb extracts on hepatoma and normal fibroblast cells using high-throughput screening in order to obtain extracts with specific anti-hepatoma effect. METHODS 242 commonly used TCM herbs were extracted by petroleum ether,ethanol and water,respectively. The total number of TCM extracts was 554. The cyto?toxicity of samples was evaluated by MTT in human hepatoma cells Bel7402 and mice normal fibroblasts NIH3T3. RESULTS 7.4%of the total extracts had an inhibitory effect greater than 50%for Bel7402,but 14.8% for fibroblasts NIH3T3 cells. Extracts with an inhibitory effect above 50% on both Bel7402 and NIH3T3 cells accounted for 4.4%of the total extracts. Our results showed that the sample DF173 had preferable cytotoxicity effect on hepatoma carcinoma cells in a good dose-effect relationship. DF173 is an ethanol extract from Stephania tetrandra,which is a commonly used herb in TCM. The cytotoxic IC50 of DF173 against Bel7402 was 8.27 mg·L-1,and 19.48 mg·L-1 on NIH3T3. CONCLUSION The components of TCM herbs are highly complicated. The combination of tumor cells with normal fibroblast cells to evaluate the cytotoxicity effect during anti-tumor drug screening will contribute much to the discovery of TCM drugs with high anti-tumor efficiency and lower toxicity.
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OBJECTIVE:To study the targeting of folic acid(FA)-modified docetaxel(DOC)nano-liposome(L-DOC-FA)to hepatocellular carcinoma Bel-7402 cells in vivo and in vitro. METHODS:The cell viability and survival rate of Bel-7402 cells was tested by CCK-8 kit after treated with 0,1,2,5,10 and 20 μg/ml DOC,L-DOC and L-DOC-FA for 24 h. And then,the fluores-cein isothiocyanate was used to label L-DOC and L-DOC-FA nano-liposome,and the rate of L-DOC and L-DOC-FA absorbed by hepatocellular carcinoma Bel-7402 cells were detected. 125I was used to label L-DOC and L-DOC-FA nano-liposome,and then the contents of them in the subcutaneous tumor tissues were detected. 28 Balb/c naked mice were selected and given liver cell suspen-sion via back ih to induce tumor model. After modeling,naked mice were divided into blank control group(normal saline),DOC group(3 mg/kg),L-DOC(3 mg/kg,by DOC)and L-DOC-FA(3 mg/kg,by DOC). They were given relevant medicine intrave-nously once a day for consecutive 30 d. The relative tumor volume in naked mice was detected. RESULTS:DOC,L-DOC and L-DOC-FA all inhibited the cell viability of Bel-7402 cells,the survival rate of cells decreased in concentration-dependant manner;compared with DOC and L-DOC,the cell viability decreased after treated with L-DOC-FA,the survival rate of cells decreased (PL-DOC (31.2%),with statistical significance (P<0.01). The content of L-DOC-FA in tumor was significantly more than that of L-DOC (P<0.01). In addition,3 mg/kg L-DOC-FA showed better inhibitory effect than 3 mg/kg L-DOC and DOC on tumor,and the rela-tive tumor volume was smaller(P<0.01). CONCLUSIONS:L-DOC-FA has obvious targeting to Bel-7402 cells in vivo and in vi-tro,and shows good inhibitory effect on tumor in vivo and in vitro.
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Objective: To investigate the antitumor activity of the compound HS-4 and the action mechanism. Methods: MTT method was used to test in vitro antitumor activity of the compound HS-4. Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice, and, in vivo antitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system. Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics, using high-throughput sequencing technology. Results: HS-4 was found to have relatively high in-vitro antitumor activity against liver cancer cells, gastric cancer cells, renal cancer cells, lung cancer cells, breast cancer cells and colon cancer cells. The IC50 values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively, while the IC50 values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L, respectively. It was demonstrated that HS-4 possessed a better therapeutic effect in liver cancer. Conclusions: A new reliable orthotopic xenotransplantation tumor model of liver cancer in nude mice is established. The new compounds HS-4 was found to possess relatively high in vivo and in vitro antitumor activity against liver cancer cells.
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OBJECTIVE@#To investigate the antitumor activity of the compound HS-4 and the action mechanism.@*METHODS@#MTT method was used to test in vitro antitumor activity of the compound HS-4. Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice, and, in vivo antitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system. Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics, using high-throughput sequencing technology.@*RESULTS@#HS-4 was found to have relatively high in-vitro antitumor activity against liver cancer cells, gastric cancer cells, renal cancer cells, lung cancer cells, breast cancer cells and colon cancer cells. The IC50 values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively, while the IC50 values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L, respectively. It was demonstrated that HS-4 possessed a better therapeutic effect in liver cancer.@*CONCLUSIONS@#A new reliable orthotopic xenotransplantation tumor model of liver cancer in nude mice is established. The new compounds HS-4 was found to possess relatively high in vivo and in vitro antitumor activity against liver cancer cells.
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Objective:To investigate the antitumor activity of the compound HS-4 and the action mechanism.Methods:MTT method was used to testin vitroantitumor activity of the compound HS-4. Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice, and,in vivoantitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system. Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics, using high-throughput sequencing technology. Results:HS-4 was found to have relatively highin-vitro antitumor activity against liver cancer cells, gastric cancer cells, renal cancer cells, lung cancer cells, breast cancer cells and colon cancer cells. The IC50 values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively, while the IC50 values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L, respectively. It was demonstrated that HS- 4 possessed a better therapeutic effect in liver cancer.Conclusions: A new reliable orthotopic xenotransplantation tumor model of liver cancer in nude mice is established. The new compounds HS-4 was found to possess relatively highin vivo andin vitroantitumor activity against liver cancer cells.
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OBJECTIVE To validate the anticancer effect of Angong Niuhuang pill (AGNH) and pinpoint associated molecular mechanisms using human cancer cells.METHODS Human MGC-803 gastric carcinoma and human BEL-7402 hepatocarcinoma cells were incubated with AGNH 9,30,90,300 and 900 mg·L-1 for 24,48and 72 h,respectively.Cell viability was detected with 3-(4,5-dimethylthiazol-2-yl) -5-( 3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and colony formation assay.Apoptosis was measured with flow cytometry and Hoechst 33258/PI staining.Change in mitochondrial membrane potential (△qψ) was detected by spectrofluorophotometer.RESULTS AGNH inhibited MGC-803 cell proliferation ( for 48 h,r =0.996,P =0.002; for72 h,r=0.756,P=0.024 ) and BEL-7402 cells (for 48 h,r =0.732,P=0.030; for72 h,r=0.702,P =0.037) in a concentration-dependent manner,as showed by MTS assay.AGNH inhibited colony formation on MGC-803 cells (r =0.914,P =0.011 ) and BEL-7402 cells (r =0.871,P =0.024) in a concentration-dependent manner for 24 h.Hoechst 33258/PI staining and flow cytometry assay showed that AGNH 900 mg·L-1 for 24 h induced apoptosis of MGC-803 and BEL-7402 cells,and the apoptosis rate was 27.2％ and 19.7％,respectively.Compared with normal control group,AGNH 900 mg·L-1 for 3 min decreased the mitochondrial membrane potential of MGC-803 and BEL-7402 cells to 15.9％ and 15.0％ of control group.CONCLUSION AGNH inhibits proliferation of human cancer cells.Apoptosis and depolarization of mitochondrial transmembrane potential are probablly its mechanism.
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Objective To identify the function and mechanism of MR1 in proliferation of BEL-7402 cells.Methods siRNA targeting MR1 and negative control siRNA were synthesized and transfected into BEL-7402 cells using Lipofectamine 2000.The silencing effect of MR1 siRNA was determined by semi-RT-PCR.SRB assay,colony formation assay and growth curve assay were used to investigate whether MR1 siRNA regulated cellular proliferation.Cell cycles were assessed by flow cytometry.G2 arrest reagent nocodazole was used to show the potential effect of MR1 siRNA on G1 arrest.The expression of Cyclin D1 was determined by Western blotting.Results(1)MR1 mRNA significantly decreased in BEL-7402 cells 24 h after MR1 siRNA transfection.(2)MR1 siRNA induced the down-regulation of cell growth.The expression of Cyclin D1 in MR1 siRNA tranfected BEL-7402 cells decreased significantly.(3)Flow cytometry results showed that MR1 siRNA markedly decreased G2 phase population with nocodazole treatment,and distinctly increased G1 phase population.Conclusion The gene MR1 is involved in the proliferation of BEL-7402 cells.MR1 siRNA causes inhibition of the proliferation of BEL-7402 cells.One of the mechanisms ofMR1 siRNA on the proliferation of BEL-7402 cells is the induction of G1 arrest.
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Objective:To investigate the effects of Bcl-2 shRNA on the growth of liver carcinoma cell line BEL-7402 and colorectal carcinoma cell line Caco2.Methods: Bcl-2 shRNA was cloned into Pgenesil-1 plasmid labeled with fluorescent protein and the product was transfected into BEL-7402 and Caco2 cells with Lipofectamine 2000.The study also included shRNA as negative control,Pgenesil-1,Lipofectamine and blank control groups.Transfected cells were visualized by inverted fluorescent microscope and then assayed by flow cytometry.The expression levels of Bcl-2 protein were assayed by Western-blot and cell proliferation was measured by MTT method.Results: There was no difference in transfection rate among cells in Bcl-2 shRNA,shRNA and Pgenesil-1 vector groups.Western-blot assay showed that the expression levels of Bcl-2 protein in BEL-7402 and Caco2 cells were significantly decreased in Bcl-2 shRNA group compared with those in other 4 groups(P
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AIM: To evaluate the inhibitory effect of galactose (Gal)-polyethyleneimine (PEI)-c-myc antisense oligodeoxynucleotide (ASODN) complex on proliferation of human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cell line Bel-7402 was treated with Gal-PEI-ASODN complex. Cell proliferation was tested by trypan blue dye at different time points and with various concentrations of ASODN treatment. Cell morphology was observed under inverted microscope, cell hypodiploid percentage was analyzed by flow cytometry and cell ultrastructure was observed through electron microscopy. RESULTS: Compared with ASODN group (20 ?mol/L) from (0 h) to 96 h, Gal-PEI-ASODN complex (with ASODN 0.75 ?mol/L) significantly suppressed Bel-7402 cells proliferation, the ASODN concentration within Gal-PEI-ASODN complex and time course acquired were significantly lower and shorter, respectively. Incubated with pure ASODN at different concentrations for 72 hours, cell proliferation was inhibited and IC_(50) was 20.9 ?mol/L; while mediated with galactose receptor for 48 hours, ASODN significantly inhibited cell proliferation and IC_(50) was only 0.294 ?mol/L, the inhibitory efficacy of ASODN enhanced 70.9 folds. While Bel-7402 cells were incubated with Gal-PEI-ASODN complex for 48 hours, cell hypodiploid percentage was much higher than ASODN groups and cell apoptosis was seen under electron microscopy. CONCLUSIONS: Galactose receptor mediated ASODN delivery may significantly increase proliferation inhibition efficacy on Bel-7402 cells. [
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AIM: To explore the different inhibitory effect of arsenic trioxide (As_2O_3) on hepatocarcinoma cell growth in SMMC-7721 and BEL-7402 cell lines and its mechanism. METHODS: The cell culture and trypan blue staining were used to study the inhibitory effect of arsenic trioxide on cell growth, and the glutathione (GSH) contents in hepatocarcinoma cells treated with arsenic trioxide were detected. RESULTS: Arsenic trioxide inhibited the growth of BEL-7402 cells in a time and dose-dependent manner. The inhibitory effect was significant at a lower dose of 0.50 ?mol/L for 24 h, however, to SMMC-7721 cells, a higher dose of 2.00 ?mol/L for 96 h was needed. The inhibitory rate of arsenic trioxide (0.25-2.00 ?mol/L) on BEL-7402 cell growth was higher than that on SMMC-7721 cells. The content of GSH in SMMC-7721 cells was much higher than that in BEL-7402 cells [(50.8?5.2) (?mol/g) protein and (18.7?1.4) ?mol/g protein, respectively]. CONCLUSION: There was a significant difference in inhibition of hepatocarcinoma cell growth by arsenic trioxide between BEL-7402 and SMMC-7721 cell lines, the cause of which may be due to the difference in GSH content in BEL-7402 and SMMC-7721 cells. [