白藜芦醇通过下调PRMT5表达抑制肝胆管癌SMMC-7721细胞的增殖、侵袭和细胞周期

目的:探究白藜芦醇(Res)通过调控PRMT5表达对肝胆管癌SMMC-7721细胞增殖、侵袭、细胞周期的影响及其机制。方法:常规培养正常肝细胞LO2和SMMC-7721细胞,用0、20、40、80μmol/L的Res进行处理,用qPCR法、MTT法、Transwell实验、流式细胞术和WB法分别检测Res处理后PRMT5 mRNA在LO2和SMMC-7721细胞中的表达,Res对SMMC-7721细胞增殖能力、侵袭能力、细胞周期和凋亡,以及PRMT5、cyclin D1和cyclin E1蛋白表达的影响。结果:PRMT5在SMMC-7721细胞中呈高表达(P<0.01);20、40、80μmol/L Res均能明显抑制PRMT5 mRNA和蛋白在SMMC-7721细胞中的表达(均P<0.01),抑制SMMC-7721细胞的增殖能力(P<0.01)和侵袭能力(P<0.05),阻滞SMMC-7721细胞周期于G0/G1期并促进其凋亡(P<0.01),明显抑制SMMC-7721细胞中周期蛋白cyclin D1、cyclin E1蛋白的表达(P<0.01)。结论:PRMT5在SMMC7721细胞中呈高表达,Res可有效抑制SMMC-7721细胞的增殖和侵袭能力并诱导其凋亡,其机制可能与抑制PRMT5表达相关。

Activity Determination of 8 Chinese Herbs against Hepatoma Cell SMMC-7721 in Vitro by MTT Method

[Objective] The aim was to build up a set of efficient and rapid models for laboratory to screen anti-hepatocellular carcinoma active substance in vitro. [ Method] By using MTT method, the activities of anti-hepatocellular carcinoma SMMC-7721 in vitro from Cymbopogon distans, Lobelia chinensis, Buddleja offlcinalis, Glycyrrhiza uralensis, Sanguisorba officinalis, Bupleurum chinense, Apium graveolen and Curuma zedoaria were tested. The growth curve of hepatoma cell was described, and the growth status in different periods were observed by inverted microscope. [ Result] Cells induced by active substance would be condensing, clear brim, which have significant differences from normal SMMC- 7721 cells. The results suggested that ESCG, ESCC, ESCB could inhibit proliferation of SMMC-7721 cells at the concentration of 1.0 -1.5 mg/ml, and the inhibition rate were 51.6%, 48.5%, 52.9% respectively. With the increasing of concentration, the inhibition strengthened. [ Conclusion] MTT method could be used as a basic model for screening important anti-hepatoma.

圣草次苷抑制肝细胞癌SMMC-7721细胞的增殖和迁移:基于激活ROS/MAPKs信号轴

目的探讨圣草次苷通过ROS/MAPKs信号轴调控肝癌SMMC-7721细胞增殖和迁移的作用机制。方法分别用0、25、50、100、150、200、250、300μg/mL圣草次苷处理肝癌SMMC-7721细胞系24 h后,CCK-8法检测细胞活性;不同浓度的圣草次苷(100、200、300μg/mL)处理细胞后,运用划痕实验分别在24、48、72 h检测划痕愈合率,Transwell实验检测细胞迁移率,克隆形成实验检测细胞增殖能力,DAPI染色观察细胞核形态变化,Western blot检测侵袭蛋白E-cadherin、N-cadherin、MMP-2、MMP-9、凋亡因子PARP和Pro-caspase 3以及MAPKs通路p-JNK、p-P38、p-ERK信号分子;用(分别为2、5、10μmol/L)ERK抑制剂U0126、JNK抑制剂SB203580和P38抑制剂SP600125预先处理细胞2 h,再用200μg/mL圣草次苷处理细胞24 h,Western blot检测凋亡蛋白PARP的切割情况;分别用N-乙酰-半胱氨酸(NAC)(30μmol/L)或圣草次苷(100、200、300μg/mL)单独或共处理细胞后,通过DCFH-DA荧光探针法分别在15、30、60 min检测内源性活性氧(ROS)水平。结果圣草次苷在50μg/mL范围内,对细胞活力基本无影响(P>0.05);圣草次苷能够显著抑制SMMC-7721细胞的划痕愈合(P<0.01),细胞迁移(P<0.01)和克隆形成(P<0.01),在300μg/mL浓度时作用最显著;圣草次苷明显减少侵袭蛋白N-cadherin、MMP-2、MMP-9的蛋白表达量(P<0.01),上调E-cadherin的蛋白表达量(P<0.05);圣草次苷诱导SMMC-7721细胞发生显著的凋亡形态学变化,Pro-caspase 3表达降低,PARP切割增多(P<0.01);圣草次苷在300μg/mL刺激30 min时ROS表达水平较高,NAC(30μmol/L)处理后,细胞内ROS表达水平下降明显;圣草次苷能够显著诱导MAPKs信号途径JNK、P38和ERK的磷酸化(P<0.01),U0126和SB203580能够增强圣草次苷诱导的PAPR切割,而SP600125逆转圣草次苷诱导的PARP切割。结论圣草次苷通过促进细胞内ROS的合成,诱导MAPKs信号通路的活化,调控肝癌细胞系SMMC-7721的增殖、迁移和凋亡。

miR-3682-3p通过调控PI3K/AKT信号通路影响SMMC-7721人肝癌细胞的增殖、侵袭和凋亡

目的分析miR-3682-3p与PI3K/AKT信号通路在肝细胞癌发展中的关系,以期为肝细胞癌的治疗提供新的思路。方法通过qRT-PCR分析MIHA人正常肝细胞和SMMC-7721人肝癌细胞中miR-3682-3p与PI3K/AKT的表达水平。通过转染不同质粒将SMMC-7721人肝癌细胞分为miR-3682-3p mimic组、miR-3682-3p mimic NC组、miR-3682-3p inhibitor组和miR-3682-3p inhibitor NC组,采用双荧光素酶报告基因实验分析miR-3682-3p与PI3K的相互作用关系。采用蛋白免疫印迹、qRT-PCR、流式细胞术、细胞划痕和Transwell实验分析不同实验组细胞的增殖、侵袭和凋亡情况。结果miR-3682-3p能够靶向调控PI3K/AKT信号通路,miR-3682-3p表达能够激活PI3K/AKT信号通路,肝癌细胞的增殖、迁移和侵袭能力增强,凋亡减少。而抑制miR-3682-3p表达后,PI3K/AKT通路受到抑制,肝癌细胞的增殖、迁移和侵袭能力减弱,凋亡增加。结论PI3K是miR-3682-3p的直接靶点,miR-3682-3p通过激活PI3K/AKT通路促进SMMC-7721人肝癌细胞的体外转移活性。

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells.METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS).RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis.CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.

Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Inhibitory activity of polysaccharide extracts from three kinds of edible fungi on proliferation of human hepatoma SMMC-7721 cell and mouse implanted S180 tumor

AIM To determine the activities ofpolysaccharide extracts from Flammulina velutipes (Curt. ex Fr. ) Sing (FV), Lentinusedodes (LE) and Agaricus bisporus Sing (AB)on the proliferation of human hepatoma SMMC-7721 cells in vitro and on mouse implanted S-180tumors in vivo.METHODS The polysaccharide extracts were isolated from the fruit bodies of FV, LE and AB by the methods of hot-water extraction, Sevag’sremoval of proteins, ethanol precipitation,trypsin digestion and ethanol fractionalprecipitation. Human hepatoma SMMC-7721 cells were treated with 50 mg/L Polysaccharide extracts, and the mitosis index, mitochondria activity and cell proliferation were detected at different times in both control and experimental groups. The mice with S-180 implanted tumors were injected with the polysaccharide extracts at 24 mg/ kg body weight for 9 d and the tumorweight was measured on the 15th day.RESULTS The mitosis index of hepatoma cells in vitro could be significantly decreased by treatment with the polysaccharide extracts fromthe three kinds of edible fungi (P < 0 .005 ). Thecell numbers and mitochondria activity of SMMC7721 cells treated with polysaccharide extracts were lower than those in control groups (P <0.005). The inhibition rates of polysaccharide extracts against implanted S-180 tumors in mice were 52.8%, 56.6% and 51 .9% respectivelycompared with that in c0ntrol gr0ups.CONCLUSI0N The POIysaccharide extractsfrom the three kinds of edible fungi could inhibitnot only the Cultured malignant cells in vitfO butalso impIanted Sl80 tum0r i0 vivo.

Positional and expressive alteration of prohibitin during the induced differentiation of human hepatocarcinoma SMMC-7721 cells

AIM： To explore the existence and distribution of prohibitin （PHB） in nuclear matrix and its co-localization with products of some related genes during the differentiation of human hepatocarcinoma SNMC-7721 cells. METHODS： The nuclear matrix of the SMMC-7721 cells cultured with or without 5 × 10^-3 mmol/L hexamethylene bisacetamide （HNBA） was selectively extracted. Western blot was used to analyze the expression of PHB in nuclear matrix; immunofluorescence microscope observation was used to analyze the distribution of PHB in cell. LCSM was used to observe the co-localization of PHB with products of oncogenes and tumor suppressor genes. RESULTS： Western blot analysis showed that PHB existed in the composition of nuclear matrix proteins and was down-regulated by HMBA treatment. Immunofluorescence observation revealed that PHB existed in the nuclear matrix, and its distribution regions and expression levels were altered after HMBA treatment. Laser scanning confocal microscopy revealed the co-localization between PHB and the products of oncogenes or tumor repression genes including c-los, c-myc, p53 and Rb and its alteration of distributive area in the cells treated by HMBA. CONCLUSION： These data confirm that PHB is a nuclear matrix protein, which is located in the nuclear matrix, and the distribution and expression of PHB and its relation with associated genes may play significant roles during the differentiation of SMMC-7721 cells.

Serum deprivation enhances DNA synthesis of human hepatoma SMMC-7721 cells

AIM To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC 7721 cells. METHODS Human hepatoma SMMC 7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf (FCS) in 5% CO 2 incubator at 37℃ for 24h , and culture media were replaced to serum free or different serum FCS levels (2 5%, 5%, 10%, 20% and 25%). Six h, 12h , 18h and 24h after the culture, the cells were incorporated TdR for 4h . At last TdR incorporation was detected with liquid scintillation counting. RESULTS DNA synthesis of SMMC 7721 cells could be sharply stimulated by short time (6h) serum deprivation (the cpm value of 3H TdR incorporation of cells in serum free was 39 32 fold higher than cells in 25% serum), and the incorporation of 3H TdR was negatively related to the serum levels. Longer time serum starvation ( 12h , 18h and 24h ) also greatly stimulated DNA synthesis, although the cpm value of 3H TdR incroporation was less than that in 6h serum deprivation. Morphology of cells cultured in different serum levels also showed significant difference. CONCLUSIONS Compared with other cell lines such as BEL7404 and Swiss 3T3, human hepatoma SMMC 7721 cells had different response to the serum deprivation. Short time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC 7721 cells. Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC 7721 cells as a model.

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells

AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.

Effects of cytotoxic T lymphocytes on hepatoma cell line SMMC-7721 induced by different subsets of dendritic cells in vitro

BACKGROUND: Dendritic cells (DCs) loaded with complex antigen are always used to induce cytotoxic T lymphocytes (CTLs) which have a specific anti-tumor activity. However, CTLs can assault autologous cells induced by DCs loaded with autologous antigen. This study aimed to explore how to weaken the autoimmune reaction induced by DC vaccine by combining mature DC (mDC) activating immunity and immature DC (imDC) leading to immune tolerance to make hepatocellular carcinoma (HCC) vaccine in vitro. METHODS: DC progenitors derived from human peripheral blood were assigned to two groups. One was cultured to mDC and pulsed with frozen-thawed antigen (FTA) of human HCC cell line SMMC-7721 cells (mDC group), and the other was cultured to imDC and pulsed with FTA of human liver cell line L-02 cells (imDC group). The morphology of DCs was monitored and cells phenotypes including HLA-DR, CD80, CD1α, CD83 were assayed by flowcytometry (FCM). The concentrations of interleukin-12 (IL-12) in the supernatant were assayed by ELISA. Methyl thiazolyl tetrazolium (MTT) was used to evaluate T cell proliferation induced by mDC and imDC and the killing rate of CTL induced by mDC and imDC respectively/together on SMMC-7721 and L-02 cells. RESULTS: Compared with the imDC group, the mDC group was characterized by the following: increased secretion of IL-12 (P【0.05); higher expression of HLA-DR, CDla, CD80, CD83; and stronger activity in stimulating proliferation of isogenic T cells (P【0.05). CTL induced by the mDC group had a significant killing response to SMMC-7721 as well as a higher killing rate for L-02 (P】0.05). CTL induced by mDC and imDC together had a higher killing response to SMMC-7721, but a lower killing rate for L-02(P【0.01). CONCLUSIONS: CTL induced by mDC and imDC together has a higher antigen-specific killing response in vitro than that induced by mDC alone. This may be of greater clinical value.

Retrovirus-mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin.The inhibitory effect of endostatin expressed by transfected 5MMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted.RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P＜0.01). In vivo experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P＜0.001).The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6±1.1, much fewer than that of 22.6±4.5 from SMMC-pLncx cells (P＜0.01).CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.

Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis

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.

Hyperthermia enhances the anticancer-drug induced cytotoxicity in hepatocellular carcinoma cell line SMMC-7721

Objective： Hyperthermia is an attractive addition to multidisciplinary approaches to clinical cancer treatment. The efficiency of hyperthermia depends on the elevation of the temperature and the duration of treatment. It has been reported that in vitro and in vivo hyperthermia enhanced the cytotoxic effect of certain anticancer drugs. However, this enhancement varies, depending on the drug used and the scheduling of treatments. Thus, the combination effect of chemotherapy and hyperthermia remains unclear. In this study, we aimed to investigate whether concurrent exposure of human hepatocellular carcinoma cells SMMC-7721 to chemotherapeutic agents andhyperthermia could increase anticancer effects. Methods ： Two chemotherapeutic agents, cisplatin and hydroxycamptothecin, were applied. The MTT assay was performed to evaluate the growth inhibition of SMMC-7721 induced by anticancer drugs with and without hyperthermia. Flow cytometric analysis was used for the assessment of apoptosis after treatments. Results： The percentages of growth inhibition of SMMC-7721 induced by cisplatin （10μg/ml） alone, hydroxycamptothecin （1μg/ml）alone, hyperthermia alone, cisplatin and hyperthermia, hydroxycamptothecin and hyperthermia, were 20.77%, 13.65%, 32.46%, 62.76%, 71.89%, respectively. The percentages of apoptosis of five treatments are 5.56%, 3.96%, 10.16%, 24.32%, 20.42%, respectively. Conclusion： While both hyperthermia and anticancer drugs can individually induce apoptosis and anti-proliferation effect, the combination of the two treatments induce significantly higher apoptosis and cytotoxicity than hyperthermia or anticancer drugs treatment alone. These data suggest a synergistic benefit when hyperthermia and anticancer drugs used concurrently.

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

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.

RNA干扰胰岛素生长因子-1受体基因对人肝癌SMMC7721增殖及凋亡蛋白的影响

目的观察RNA干扰胰岛素生长因子-1(IGF-1)受体基因后对人肝癌细胞株SMMC7721增殖和相关凋亡因子表达的影响。方法构建两个靶向IGF-1R基因的RNA干扰真核表达质粒(IGF-1R-siRNA-1和IGF-1R-siRNA-2)分别转染至SMMC7721细胞,并设立正常对照组、空白对照组、IGF-1R-siRNA-1转染组、IGF-1R-siRNA-2转染组。采用聚合酶链反应(RT-PCR)法和Western blot检测IGF-1R基因表达变化,检测细胞增殖、侵袭、凋亡、凋亡蛋白、骨形态发生蛋白(BMPs)及相关信号通路蛋白的表达水平。结果RT-PCR结果显示,IGF-1R-siRNA-1和IGF-1R-siRNA-2表达载体转染SMMC-7721细胞后,IGF-1R基因的mRNA水平显著下调,IGF-1R-siRNA-1转染组IGF-1R基因的表达低于正常对照组,IGF-1R-siRNA-2转染组IGF-1R基因表达下调77.5%,差异有统计学意义(P<0.05)。Western blot结果显示,IGF-1R-siRNA-1转染组和IGF-1R-siRNA-2转染组IGF-1R蛋白在不同水平显著下调,IGF-1R蛋白表达抑制率分别为73.0%和81.5%,差异有统计学意义(P<0.05)。IGF-1R-siRNA-1转染组和IGF-1R-siRNA-2转染组细胞增殖率明显低于空白对照组,差异有统计学意义(P<0.05)。ImageJ软件分析划痕面积结果显示,与正常对照组和空白对照组相比,IGF-1R-siRNA-1转染组、IGF-1R-siRNA-2转染组修复率下降,差异有统计学意义(P<0.05)。Transwell实验发现,与正常对照组和空白对照组相比,IGF-1R-siRNA-1转染组、IGF-1R-siRNA-2转染组为SMMC-7721细胞侵袭能力明显下降,侵袭细胞数量明显减少,差异有统计意义(P<0.05)。流式细胞检测显示,IGF-1R-siRNA-1转染组和IGF-1R-siRNA-2转染组晚期凋亡率明显高于空白对照组,差异有统计学意义(P<0.05)。Western blot结果显示,IGF-1R-siRNA-1组、IGF-1R-siRNA-2组Bcl-2表达均明显受抑制,Bax、Cleaved Caspase-3蛋白表达量明显高于空白对照组,差异有统计学意义(P<0.05);PI3K、AKT/p-AKT、BMP-2、BMP-7蛋白表达量明显低于空白对照组,差异有统计学意义(P<0.05)。结论由IGF-1R基因沉默对肝癌SMMC7721细胞增殖与侵袭有抑制作用,对细胞凋亡有促进作用,能介导BMP2、BMP-7基因不同水平下调,IGF-1/PI3K/AKT信号通路蛋白可能参与其中。

Enhanced radioimmunotherapeutic efficacy of a monoclonal antibody cocktail against SMMC-7721 human hepatocellular carcinoma

The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal atibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. boioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I labeled Hepama-1 and 131Llabeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 Mab alone The MAb cocktail could target a greater number of hepstoma cells and increase the magnitude of hepatoma cen uptde of radioamibodies. The in vjtro results explain the enhanced effect of the MAb cocktail in in vjvo model system.

rAdinbitor, a novel disintegrin from Agkistrodon halys brevicaudus stejneger inhibits adhesion and proliferation of SMMC-7721 cells

Objective： To investigate the effects of rAdinbitor on the adhesion and proliferation of human hepatoma cell strain 8MMC-7721. Methods： Cell adhesion assay was used to observe the effect of rAdinbitor on the adhesion of 8MMC-7721 cells to fibronectin （FN）. Crystal violet staining was performed to detect the influence of rAdinbitor on the adhesion of 8MMC- 7721 cells. MTT assay was employed to detect the inhibitory effects of different concentration of rAdinbitor on the proliferation of 8MMC-7721 cells. The morphologic changes of the control 8MMC-7721 cells and the apoptotic cells induced by 200μg/mL rAdinbitor for 36 h were observed under light microscope after HE staining. Flow cytometry analysis was applied to determine the apoptosis rate of 8MMC-7721 cells. Results： （1） FN promoted the adhesion of human hepatoma cell strain 8MMC-7721 in a dose-dependent manner. （2） rAdinbitor could dose-dependently inhibit the adhesion of SMMC-7721 cells to FN. The higher the concentration was, the stronger the inhibition was. There was significant difference among the groups （P 〈 0.05）. （3） rAdinbitor had a strong inhibition on the proliferation of 8MMC-7721 cells and showed a dose-dependent manner （P 〈 0.05）. After a 48 h exposure, the IC50 value of rAdinbitor was 177.83 μg/mL. （4） After exposure of 8MMC-7721 cells to 200μg/mL rAdinbitor for 36 h, the early morphologic changes appeared and the apoptosis rate was 20.68%, significantly higher than that of the control group （2.38%, P 〈 0.05）. Conclusion： rAdinbitor can dose-dependently inhibit the 8MMC-7721 cells adhesion to FN, and can inhibit the proliferation in dose-dependent manner and promote their apoptosis.

Combined anti-tumor effects of mDRA-6 and nimesulide on human hepatocellular cancer cell line SMMC-7721

Objective: The aim of this work was to evaluate anti-tumor effects of mDRA-6 plus nimesulide on a human hepatocellular cancer cell line, SMMC-7721, and study the main mechanisms. Methods: The DR5 receptor of SMMC-7721 cells was detected by flow cytometry (FCM). For further experimental application, SMMC-7721 cells were treated with proper dose of mDRA-6, nimesulide, or mDRA-6 plus 200 μmol/L nimesulide; untreated SMMC-7721 cells were comparably set as control. Cytotoxicity was tested by MTT assay; cell morphology was examined using Hoechst 33258 staining; and apoptosis was determined by FCM. Results: The positive rate of DR5 on SMMC-7721 was 95.0%. Either mDRA-6 or nimesulide alone induces SMMC-7721 cell death in a dose-dependent manner. Treatment of 1,600 ng/mL mDRA-6 for 12h led to a cell-death rate of 35.0%, while an increased cell-death rate (91.1%) was found under the same condition of mDRA-6 treatment supple- mented with 200 μmol/L nimesulide. Hoechst 33258 and Annexin V/PI staining confirmed apoptosis as the main cause of this anti-tumor response. Conclusion: Both mDRA-6 and nimesulide can induce apoptosis of SMMC-7721 cells, and they have synergistic anti-tumor activities against SMMC-7721.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.