Synthesis of matrine derivative ZS10 and its mechanism of apoptosis induced by PI3K/AKT signaling pathway in human hepatocellular carcinoma cell line BEL-7402 / 中国药理学通报

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.

Tertiary sulphonamide derivatives as dual acting small molecules that inhibit LSD1 and suppress tubulin polymerisation against liver cancer.

A series of tertiary sulphonamide derivatives were synthesised and evaluated for their antiproliferative activity against liver cancer cell lines (SNU-475, HepG-2, and Bel-7402). Among these tertiary sulphonamides, compound 17a displayed the best anti-liver cancer activity against Bel-7402 cells with an IC50 value of 0.32 µM. Compound 17a could effectively inhibit tubulin polymerisation with an IC50 value of 1.27 µM. Meanwhile, it selectively suppressed LSD1 with an IC50 value of 63 nM. It also concentration-dependently inhibited migration against Bel-7402 cells. Importantly, tertiary sulphonamide 17a exhibited the potent antitumor activity in vivo. All these findings revealed that compound 17a might be a tertiary sulphonamide-based dual inhibitor of tubulin polymerisation and LSD1 to treat liver cancer.

Inhibitory effect of taspine derivative TAD1822-7 on tumor cell growth and angiogenesis via suppression of EphrinB2 and related signaling pathways.

The aim of this study was to investigate the inhibitory effect of TAD1822-7, a synthesized taspine derivative, on cancer through its effects on tumor cell growth and angiogenesis via suppression of EphrinB2. The obtained data showed that TAD1822-7 decreased Bel-7402 cell viability and colony formation ability and suppressed cell migration. TAD1822-7 effectively inhibited blood vessel formation in an aortic ring assay to examine angiogenesis. Moreover, it also down regulated the expression of VEGFR2, VEGFR3, CD34, PLCγ, Akt, MMP2, MMP9, and CXCR4, and suppressed the expression of EphrinB2 and its PDZ protein, PICK1, in Bel-7402 cells. These results indicate that TAD1822-7 is a potential anti-angiogenic agent that can inhibit the viability and migration of Bel-7402 cells via suppression of EphrinB2 and the related signaling pathways.

Evodiamine induces apoptosis of human hepatoma cell line BEL-7402 by Hippo-YAP pathway / 中草药

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.

Tegillarca granosa extract Haishengsu inhibits tumor activity via a mitochondrial­mediated apoptotic pathway.

Haishengsu (HSS) is an active natural extract isolated from Tegillarca granosa, which has previously been demonstrated to inhibit the proliferation of several types of cancer cells in vitro. Our previous study indicated that HSS may induce apoptosis to suppress growth of human hepatocellular carcinoma BEL­7402 cells by activating Fas pathway. The present study demonstrated that HSS treatment induces the in vitro apoptosis of BEL­7402 cells via the mitochondrial­mediated apoptotic pathway detected by DNA fragmentation assay, caspase activity assay and transmission electron microscopy assay, and inhibits tumor xenograft growth in vivo. Alterations in apoptotic regulatory proteins were detected, including decreased expression of B­cell lymphoma2 (Bcl­2), upregulation of Bcl­2­associated X protein and mitochondrial cytochrome c release, and downstream activation of apoptotic signaling. Furthermore, apoptotic induction was caspase­dependent, as indicated by cleavage of the caspase substrate, poly (ADP­ribose) polymerase. Oral administration of 62.5­250 mg/kg HSS markedly educed the growth of hepatocellular carcinoma tumor xenografts in nude mice. In addition, immunohistochemical staining for caspase­3 protein and transmission electron microscopy further indicated the induction of apoptosis in these tumor tissues. Taken together, the present study demonstrated that HSS may effectively induce apoptosis to suppress the growth of BEL­7402 cells in vitro and in vivo, and therefore may hold promise for further development as a novel cancer therapy.

[Differences of Therapeutic Efficacy Between Different Kinds of Somatostatin Analogue for Primary Hepatocellular Carcinoma].

OBJECTIVE: To explore the effects of SOM230, octreotide and lanreotide on hepatocellular carcinoma cell line Bel-7402 In vivoand In vitro, and to analyze the differences of their therapeutic efficacy with relevant mechanisms. METHODS: At different time points (24, 48, 72 h), the cell counting kit-8 (CCK8) was used to evaluate cell proliferation (drug concentration 1×10-10-1×10-5mol/L) and the Annexin Ⅴ-FITC/PI staining was used to assess cell apoptosis (drug concentration 1×10-5mol/L), while the real-time quantitative PCR was used to detect cellular SSTRexpression changes before and after interventions. A transplanted tumor model was set up, and the tumor-bearing nude mice were treated by three drugs with a common dose of 100 µg/ (kg·d) or same volume of normal saline, respectively. After a treatment period of 6 weeks, real-time quantitative PCR and Western blot test were performed to detect the expression changes ofSSTR in tumor tissues from the level of gene and protein. RESULTS: All three drugs could inhibit the cell proliferation of Bel-7402. However, they were unable to promote the cell apoptosis. In vitro, the expressions ofSSTR1, SSTR4genes did not change over time in each group. The expressions ofSSTR2gene were decreased in three intervention groups while the expressions ofSSTR5gene were increased first and then decreased. Compared with the control group, all differences have statistical significance (P<0.05). All three drugs could improve the survival rate and quality of life for nude mice bearing hepatoma. In vivo, the expression ofSSTR1gene in SOM230 group was increased when compared with that of the other groups; the expressions ofSSTR2gene in three intervention groups were increased when compared with that of the control group; the expressions of SSTR5gene in SOM230 group and lanreotide group were increased when compared with that of the octreotide group and the control group, and all differences have statistical significance (P<0.05). The Western blot test confirmed these results from the protein level. CONCLUSION: In a certain concentration range, the long-term treatment of SSTA with high affinities to SSTR2 and SSTR5 could inhibit the growth of HCC.

MiR-503 sensitizes human hepatocellular carcinoma cells to cisplatin by targeting bcl-2 / 中南大学学报(医学版)

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.

Study on Targeting of Folic Acid-modified Docetaxel Nano-liposome to Hepatocellular Carcinoma Bel-7402 Cells in vivo and in vitro / 中国药房

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.

High-throughput screening of single Chinese plant extracts on specific anti-hepatoma drugs / 中国药理学与毒理学杂志

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.

HS-4, a highly potent inhibitor of cell proliferation of human cancer cell.

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.

Optimization of processing technology of Rhizoma Pinelliae Praeparatum and its anti-tumor effect.

BACKGROUND: Rhizoma Pinelliae is the dried tuber of Pinellia ternata (Thunb.) Breit. Modern pharmacological studies have shown that Rhizoma Pinelliae has antitussive, antiemetic, glandular secretion inhibiting and antitumor effects. OBJECTIVES: To optimize the processing technology of Rhizoma Pinelliae Praeparatum, and to study its anti-tumor effect. METHODS: Orthogonal design method was applied to analyze the effects of factors such as licorice concentration volume, soaking time and processing temperature on processing technology of Rhizoma Pinelliae Praeparatum; MTT assay and flow cytometry were used to determine the inhibitory effect of Rhizoma Pinelliae Praeparatum on Bel-7402 cells. RESULTS: During the processing of Rhizoma Pinelliae Praeparatum, the size of influence of licorice concentration volume, soaking time and processing temperature on processing results of Rhizoma Pinelliae was: B>C>A in descending order, i.e. soaking time>processing temperature>licorice concentration volume, different concentrations of Rhizoma Pinelliae Praeparatum ethanol extracts could all exert inhibitory effect on the growth and proliferation of Bel-7402 cells, and with the increase of drug concentration and the extension of culture time, the cell proliferation inhibitory effect of Rhizoma Pinelliae Praeparatum ethanol extract became more and more evident. Apoptotic rate of 1.5 mg/ml Rhizoma Pinelliae Praeparatum ethanol extract group reached 13.53%, the difference was extremely significant compared with the control group. In conclusion the factor most influential to the processing technology of Rhizoma Pinelliae Praeparatum was soaking time, followed by processing temperature, the factor least influential was licorice concentration volume. CONCLUSION: Rhizoma Pinelliae Praeparatum has inhibitory effect on growth and proliferation of Bel-7402 cells.

Anti-tumor effect of α-pinene on human hepatoma cell lines through inducing G2/M cell cycle arrest.

Pine needle oil from crude extract of pine needles has been used as an anti-cancer agent in Traditional Chinese Medicine. The α-pinene is a natural compound isolated from pine needle oil which has been shown anti-cancer activity. In previous study, we found that pine needle oil exhibited significant inhibitory effect on hepatoma carcinoma BEL-7402 cells. In this study, we investigate the inhibition of α-pinene on hepatoma carcinoma BEL-7402 cells in vitro and in vivo and further explore the mechanism. The results show that liver cancer cell growth was inhibited obviously with inhibitory rate of 79.3% in vitro and 69.1% in vivo, Chk1 and Chk2 levels were upregulated, CyclinB, CDC25 and CDK1 levels were downregulated.

HS-4, a highly potent inhibitor of cell proliferation of human cancer cell

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.

HS-4, a highly potent inhibitor of cell proliferation of human cancer cell

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.

HS-4, a highly potent inhibitor of cell proliferation of human cancer cell

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.

Differentiation of bel-7402 human hepatocarcinoma cells induced by aqueous extracts of fresh gecko (AG) and its anti-tumor activity in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Gecko, a kind of reptile, has been widely used as a traditional Chinese medicine to treat various diseases including cancer in China for thousands of years. The aim of this study was to investigate the anti-tumor effect of AG (aqueous extracts of fresh gecko) on human hepatocellular carcinoma cell Bel-7402 in vitro and mouse H22 hepatocellular in vivo. Further to underlie the molecular mechanism of AG inducing the differentiation of Bel-7402 cells. MATERIALS AND METHODS: AG was obtained by water extracting method and qualitatively analyzed through High Performance Liquid Chromatography. The total protein concentration of AG was measured by BCA (bicinchoninic acid disodium) assay. The anti-tumor activities in vivo were analyzed through H22 (mouse hepatocellular carcinoma cell line H22) tumor xenografts mice. The cytotoxic activity of AG on Bel-7402 cells was evaluated by MTT assays. AFP (alpha fetoprotein) was detected by radioimmunoassay. ALB (albumin), ALP (alkaline phosphatase) and γ-GT (γ-glutamyl transpeptidase) were detected by biochemical methods with commercial kits. While morphological changes were observed through an inverted microscope. Moreover, the expression level of the proteins involved in MAPK (mitogen-activated protein kinase) signal pathway which was closely related to cellular differentiation was assessed by Western blot. RESULTS: AG showed obviously anti-tumor activity in vivo and anti-proliferative activity on Bel-7402 cells in vitro both dose-dependently. The number of clones of Bel-7402 cells treated with AG reduced and the cells were displaying differentiation state such as relatively bigger size and dispersed growth. The biochemical function markers of the cells were significantly changed after being treated with AG. The data showed that AFP secretion of the cells decreased 42.5%, ALB secretion increased 58.9%, the activity of ALP and γ-GT markedly decreased 67.0% and 48.5% separately when the concentration of AG was 10µl/ml, and those effects were all in a dose-dependent manner. The major original and phosphorylated signal proteins (ERK1/2 (extracellular sigal-regualted kinase 1/2), P38 (p38 MAPK) and JNK1/2 (c-Jun N-terminal kinase 1/2)) involved in MAPK signal pathway were measured and the results showed that AG activated the ERK1/2 of Bel-7402 cells. CONCLUSIONS: AG has anti-tumor activity in vivo and inhibits Bel-7402 cell proliferation in vitro through inducing cell differentiation, and the mechanism involves the activation of ERK1/2.

Anti-proliferation of Angong Niuhuang pill on tumor cells via inducement of apoptosis and down-regulation of mitochondrial membrane potential / 中国药理学与毒理学杂志

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.

MR1 siRNA suppresses proliferation of human hepatoma BEL-7402 cells / 基础医学与临床

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.

Inhibitory effect of Bcl-2 short hairpin RNA on growth of BEL-7402 and Caco2 cells / 第二军医大学学报

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

Inhibition of human hepatocellular carcinoma cell proliferation by galactose receptor-mediated c-myc antisense oligonucleotide / 中国病理生理杂志

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. [