AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 c...AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.展开更多
Background Ursolic acid （UA） is a ubiquitous molecule in the plant kingdom with specific anticancer effects that have been shown in vitro and in vivo. Although UA can inhibit the proliferation of liver cancer cells ...Background Ursolic acid （UA） is a ubiquitous molecule in the plant kingdom with specific anticancer effects that have been shown in vitro and in vivo. Although UA can inhibit the proliferation of liver cancer cells and induce apoptosis of many types of tumor cells, the molecular mechanism of its anti-hepatoma activity is still not well defined. The objective of this study was to investigate the inhibitory effect and mechanisms of UA on the human hepatoma cell line SMMC-7721. Methods After treatment with UA, the growth inhibition of SMMC-7721 cells was assessed by MTT assay. Cells were also evaluated by flow cytometric analysis, Wright-Giemasa staining, Hoechst 33258 staining and transmission electron microscope after they were induced by UA. DNA microarray technology was used to investigate the gene expression pattern of SMMC-7721 cells exposed to UA 40 pmol/L. The molecular mechanism of cells death was analyzed by real-time RT-PCR and Western blotting. Results The proliferation of SMMC-7721 cells was significantly inhibited in a dose- and time-dependent manner after UA treatment. UA induced cell cycle arrest and apoptosis. The DNA microarray analysis indicated that 64 genes were found to be markedly up- or down-expressed, including GDF15, SOD2, ATF3, and fos. The result of Western blotting showed the apoptotic proteins p53 and Bax were up-regulated while the anti-apoptotic protein Bcl-2 was down-regulated. Real-time RT-PCR confirmed UA could up-regulate the mRNA expressions of GDF15, SOD2, ATF3 and down-regulate the mRAN expression of fos. Meanwhile these effects were partly blocked by pretreatment with the p53 inhibitor Pft-a. Conclusion Activation of the p53 pathway is involved in UA inhibition of SMMC-7721 human hepatocellular carcinoma cell growth and induction of apoptosis.展开更多
AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepato...AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7,-9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation ofhuman hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.展开更多
AIM： To investigate the cyclooxygenase-2 （COX-2） expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth in...AIM： To investigate the cyclooxygenase-2 （COX-2） expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis. METHODS： Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization （ISH） and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-（4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium （MTT） bromide colorimetric assay. Celecoxib- induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） and flow cytometry （FCM）. The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis. RESULTS： Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%,and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt （Thr^308）. Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt （Thr^308） after treatment with celecoxib. CONCLUSION： COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.展开更多
目的:研究白花蛇舌草豆甾醇(stigmasterol from Hedyotis diffusa willd.,SHD)对人肝癌细胞SMMC-7721、BEL-7402的体外抑制作用,对肝癌H22的体内抑制作用及对其增殖周期、凋亡的影响。方法:MTT法评价SHD对人肝癌细胞SMMC-7721、BEL-740...目的:研究白花蛇舌草豆甾醇(stigmasterol from Hedyotis diffusa willd.,SHD)对人肝癌细胞SMMC-7721、BEL-7402的体外抑制作用,对肝癌H22的体内抑制作用及对其增殖周期、凋亡的影响。方法:MTT法评价SHD对人肝癌细胞SMMC-7721、BEL-7402的抑制率变化规律。昆明雄性小鼠60只,随机取10只为正常对照组,余接种H22瘤株,随机分为模型对照组、5-FU阳性对照组(30mg/kg)和高中低剂量SHD给药组(剂量分别为15、30、60mg/kg),腹腔给药10 d后,比较各组瘤重抑制率、H22细胞周期分布、凋亡率。结果:SHD对SMMC-7721、BEL-7402细胞具有体外抑制作用;SHD显著抑制H22肿瘤,增加G0-G1期细胞比例,降低G2/M期细胞比例,促进肿瘤细胞凋亡。结论:SHD在体外、体内均具有抑制肝癌细胞的作用,此作用与阻滞肿瘤细胞增殖周期,促进肿瘤细胞凋亡有关。展开更多
文摘AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.
文摘Background Ursolic acid （UA） is a ubiquitous molecule in the plant kingdom with specific anticancer effects that have been shown in vitro and in vivo. Although UA can inhibit the proliferation of liver cancer cells and induce apoptosis of many types of tumor cells, the molecular mechanism of its anti-hepatoma activity is still not well defined. The objective of this study was to investigate the inhibitory effect and mechanisms of UA on the human hepatoma cell line SMMC-7721. Methods After treatment with UA, the growth inhibition of SMMC-7721 cells was assessed by MTT assay. Cells were also evaluated by flow cytometric analysis, Wright-Giemasa staining, Hoechst 33258 staining and transmission electron microscope after they were induced by UA. DNA microarray technology was used to investigate the gene expression pattern of SMMC-7721 cells exposed to UA 40 pmol/L. The molecular mechanism of cells death was analyzed by real-time RT-PCR and Western blotting. Results The proliferation of SMMC-7721 cells was significantly inhibited in a dose- and time-dependent manner after UA treatment. UA induced cell cycle arrest and apoptosis. The DNA microarray analysis indicated that 64 genes were found to be markedly up- or down-expressed, including GDF15, SOD2, ATF3, and fos. The result of Western blotting showed the apoptotic proteins p53 and Bax were up-regulated while the anti-apoptotic protein Bcl-2 was down-regulated. Real-time RT-PCR confirmed UA could up-regulate the mRNA expressions of GDF15, SOD2, ATF3 and down-regulate the mRAN expression of fos. Meanwhile these effects were partly blocked by pretreatment with the p53 inhibitor Pft-a. Conclusion Activation of the p53 pathway is involved in UA inhibition of SMMC-7721 human hepatocellular carcinoma cell growth and induction of apoptosis.
基金Supported by the National Key Technologies Research and Development Program of China during the 10th Five-Year Plan Period,No.2002BA760C
文摘AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7,-9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation ofhuman hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.
基金Supported by Medical Science Research Foundation of Jiangsu Health Bureau Grant Z200314 (to JL)Medical Science Research Foundation of Nanjing Medical University Grant NY1999023 (to NBL) and CX2003012 (to JL)
文摘AIM： To investigate the cyclooxygenase-2 （COX-2） expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis. METHODS： Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization （ISH） and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-（4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium （MTT） bromide colorimetric assay. Celecoxib- induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） and flow cytometry （FCM）. The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis. RESULTS： Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%,and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt （Thr^308）. Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt （Thr^308） after treatment with celecoxib. CONCLUSION： COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.
