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.

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.

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.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

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.

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.

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.

香加皮提取物杠柳苷抑制SMMC-7721细胞Stat3信号通路诱导细胞凋亡的研究

目的研究香加皮提取物杠柳苷对Stat3信号通路的影响,及其诱导细胞凋亡的分子机制。方法MTT比色法检测人肝癌细胞SMMC-7721增殖活性,流式细胞术分析肿瘤细胞的周期变化和凋亡,Western blot检测细胞内Stat3蛋白表达,RT-PCR检测细胞Mcl-1、Survivin和XIAP mRNA的表达。结果杠柳苷可明显抑制SMMC-7721细胞的增殖,诱导SMMC-7721细胞凋亡,并使细胞周期停滞在G2/M期。杠柳苷作用后,细胞核内Stat3量降低,而细胞质中的量未见明显改变,细胞内Mcl-1、Survivin和XIAP基因的表达明显降低,并呈时间依赖性。结论香加皮提取物杠柳苷通过抑制Stat3信号转导通路和Mcl-1、Survivin和XIAP mRNA的表达,进而抑制SMMC-7721细胞的增殖,诱导其凋亡。

香加皮杠柳苷抑制Stat5信号通路诱导SMMC-7721细胞凋亡的实验研究

目的研究香加皮提取物杠柳苷对Stat5信号通路的影响,探讨其诱导肿瘤细胞凋亡的分子机制。方法MTT比色法检测细胞增殖活性,流式细胞术观察肿瘤细胞的周期变化和凋亡,western blot法检测杠柳苷作用前后人肝癌细胞株SMMC-7721细胞内Stat5蛋白质表达。结果杠柳苷明显抑制SMMC-7721细胞的增殖,使细胞周期停滞在G2/M期,并能够诱导SMMC-7721细胞凋亡。杠柳苷作用后,细胞核内的Stat5蛋白量降低而胞浆中的量未见明显改变。结论香加皮杠柳苷能够抑制Stat5信号转导通路,进而抑制SMMC-7721细胞的增殖并诱导其凋亡。

β-谷甾醇、豆甾醇诱导人肝癌细胞SMMC-7721凋亡

目的研究β-谷甾醇、豆甾醇诱导人肝癌细胞SMMC-7721的凋亡作用。方法 MTT法观察β-谷甾醇、豆甾醇对细胞增殖的抑制作用;激光共聚焦显微镜观察细胞形态变化;用流式细胞仪检测细胞周期、凋亡率、细胞内活性氧ROS、钙离子Ca2+含量、线粒体膜电位ΔΨm变化。结果β-谷甾醇、豆甾醇均明显抑制SMMC-7721细胞增殖,使细胞形态发生典型凋亡变化,凋亡率和细胞内Ca2+、ROS的含量均显著增加,线粒体膜电位降低。β-谷甾醇使细胞周期阻滞在G2/M期,而豆甾醇则同时阻滞在S期和G2/M期。结论β-谷甾醇、豆甾醇具有抑制人肝癌细胞SMMC-7721的增殖和诱导细胞凋亡的作用。

奥沙利铂和人参皂苷Rg3不同联合方式对人肝癌细胞株SMMC-7721作用的影响

目的通过体外实验观察奥沙利铂和人参皂苷Rg3不同联合方式对人肝癌细胞株SMMC-7721作用的影响,并初步探讨其机制。方法采用MTT法观察奥沙利铂、人参皂苷Rg3单药以及两药同时和序贯应用对SMMC-7721细胞增殖的作用;应用流式细胞术分析单药和两药不同序贯方式对细胞周期分布及凋亡的影响;Western blotting检测单药和两药不同序贯方式作用后SMMC-7721细胞cyclin D1蛋白的表达情况。结果奥沙利铂、人参皂苷Rg3单药、两药同时及序贯给药对SMMC-7721细胞增殖均有明显的抑制作用。同时用药组的增殖抑制作用优于人参皂苷Rg3先用组(P<0.05),但与奥沙利铂先用组比较未见明显差异(P>0.05);奥沙利铂先用组的效果优于人参皂苷Rg3先用组(P<0.05)。流式细胞术分析显示,奥沙利铂主要将细胞阻滞在S期、G2/M期,人参皂苷Rg3主要将细胞阻滞在G0/G1期,同时用药组和奥沙利铂先用组的凋亡率相似(P>0.05),阻滞细胞于G2/M期,且凋亡率均较先用人参皂苷Rg3组高(P<0.05)。Western blotting显示,两药序贯及同时应用组cyclin D1蛋白表达明显低于单药组,奥沙利铂先用组与同时用药组相似,均低于人参皂苷Rg3先用组。结论奥沙利铂、人参皂苷Rg3序贯及同时应用较单药更能抑制人肝癌SMMC-7721细胞的增殖,先用奥沙利铂后序贯人参皂苷Rg3与两药同时应用的协同增效作用相似,均优于先用人参皂苷Rg3后序贯奥沙利铂,其机制可能与奥沙利铂先用组和同时用药组将细胞阻滞在S期、G2/M期,增加促凋亡作用,同时下调cyclin D1蛋白的表达水平有关。

龙胆苦苷等6种中草药提取物对SMMC-7721人肝癌细胞增殖的影响

目的 研究龙胆苦苷等 6种中药有效成分对 SMMC- 772 1人肝癌细胞增殖的影响。方法 MTT法测定不同浓度的 SMMC- 772 1人肝癌细胞活力 ,观察药物对癌细胞增殖的影响。结果 10 2 ,10 4和 10 6nmol.L-1的土贝母皂苷、10 4nmol.L-1和 10 2 nmol.L-1的龙胆苦苷、10 2 nmol.L-1的白屈菜红碱、 3个不同浓度的血根碱及 1g.L-1的败酱草提取物作用于细胞 4 8h后对细胞具有明显的杀伤效应 ;10 mg.L-1的败酱草提取物和 1g.L-1和 10 mg.L-1的威灵仙提取物对肝癌细胞具有促生长作用。结论 龙胆苦苷、土贝母皂苷、白屈菜红碱和血根碱能抑制 SMMC- 772 1人肝癌细胞增殖 ;败酱草提取物能促进肝癌细胞增殖 ;低浓度威灵仙提取物抑制、高浓度促进肝癌细胞增殖。

水红花子黄酮类成分对人肝癌细胞株SMMC-7721的影响

目的:研究水红花子黄酮类成分对人肝癌细胞株SMMC-7721增殖、细胞周期及细胞凋亡的影响。方法:采用MTT法研究水红花子黄酮类成分对人肝癌细胞SMMC-7721的抑制作用及时效量效关系;并通过PI染色法和Annexin V-EGFP/PI双染法观察水红花子黄酮类成分作用于人肝癌细胞株SMMC-7721后细胞周期DNA含量变化及细胞凋亡率。结果:水红花子黄酮类成分能调节SMMC-7721人肝癌细胞株G1/S转换,使肿瘤细胞发生S期阻滞,造成S期细胞堆积,阻断细胞的DNA合成和复制,阻滞肿瘤细胞进入G2/M期,从而抑制肿瘤细胞的增殖,诱导SMMC-7721细胞凋亡,且具有明显的时效及量效关系。结论:水红花子黄酮类成分对人肝癌细胞株SMMC-7721具有明显的抑制作用,且抑制作用和时间剂量成线性关系,其作用机制为抑制肿瘤细胞的增殖和诱导该细胞的凋亡。

白茅根水提物对人肝癌细胞株SMMC-7721细胞周期及细胞凋亡的影响

目的研究白茅根水提物对人肝癌细胞株SMMC-7721增殖抑制率、细胞周期及细胞凋亡的影响。方法采用MTT法研究白茅根水提物对人肝癌细胞SMMC-7721的抑制率及抑制率与时间剂量关系;并通过PI染色法和Annexin V-EGFP/PI双染法研究白茅根水提物作用于人肝癌细胞株SMMC-7721后细胞周期DNA含量变化及对细胞凋亡率的影响。结果不同浓度的白茅根水提物处理肝癌SMMC-7721细胞24,48,72,96 h后,均呈现出显著的细胞增殖抑制作用,抑制作用具有明显的时间-剂量-效应关系,差异有统计学意义(P<0.05);细胞周期与细胞凋亡实验结果显示不同浓度的白茅根水提物处理肝癌SMMC-7721细胞46 h后可显著增加肝癌细胞凋亡率,差异有统计学意义(P<0.001),作用36 h后,可增加S期比例同时降低G2/M期细胞比例,差异有统计学意义(P<0.001)。结论白茅根水提物对人肝癌细胞株SMMC-7721具有明显的增殖抑制作用并可诱导其凋亡,该作用是通过抑制G2/M期细胞比例,将细胞周期阻滞在S期实现的。

STAT5在人肝癌细胞株SMMC-7721细胞内的表达

目的研究信号传导及转录活化因子STAT5在人肝癌细胞内的表达及其酪氨酸磷酸化活化情况。方法分别采用western b lot、流式细胞术(FCM)方法检测人肝癌细胞株SMMC-7721细胞内STAT 5蛋白和磷酸化STAT 5(P-STAT5)蛋白的表达。结果人肝癌细胞株SMMC-7721细胞内有STAT 5的表达和酪氨酸(Tyr 694)磷酸化活化。结论STAT 5的异常表达和活化可能在肝癌的发生发展中起重要的作用。

华蟾素诱导人肝癌SMMC-7721细胞凋亡的实验研究

目的:探讨华蟾素诱导人肝癌细胞凋亡及作用机理。方法:采用 MTT 法、AO/EB 荧光染色法、流式细胞仪及免疫组化等方法,观察华蟾素对人肝癌 SMMC-7721细胞株的作用。结果:华蟾素可显著抑制SMMC-7721细胞的生长;经药物处理后,AO/EB 染色时,肝癌细胞在显微镜下呈现出典型的凋亡形态学改变;在流式细胞仪上可见华蟾素能将细胞周期阻断在 S 期;对抗凋亡基因 bcl-2表达有一定的抑制作用。结论:华蟾素能抑制人肝癌 SMMC-7721细胞生长;能诱导人肝癌细胞凋亡;其诱导细胞凋亡的机理与其能阻断细胞周期、抑制 bcl-2基因表达有关。

STAT3在人肝癌细胞株SMMC-7721细胞内的表达

目的研究信号传导及转录活化因子STAT3在人肝癌细胞内的表达及其表达产物STAT3酪氨酸磷酸化活化情况。方法分别采用western blotting、流式细胞术检测人肝癌细胞株SMMC-7721细胞内STAT3蛋白和磷酸化STAT3(P-STAT3)蛋白的表达。结果人肝癌细胞株SMMC-7721细胞内有STAT3的表达和酪氨酸(Y705)磷酸化。结论STAT3的异常表达和活化可能在肝癌的发生发展中起重要作用。

黄芩苷对肝癌细胞株SMMC-7721裸鼠移植瘤生长抑制作用及其机制

目的:以裸鼠人肝癌细胞株SMMC-7721移植瘤动物模型为研究对象,观察黄芩苷对移植瘤生长能力的影响,初步探讨黄芩苷对Cyclin D1和Caspase-3表达的影响。方法:BALB/c裸鼠右前腋部皮下接种人肝癌SMMC-7721细胞5×106个,建立人肝癌实体瘤模型。将20只成瘤裸小鼠随机分为空白对照组与黄芩苷组,每组10只。对照组腹腔注射生理盐水10ml/kg,黄芩苷组腹腔注射黄芩苷注射液100mg/kg,每2d给药1次,间断给药8次后称体重、处死,瘤块离体称重,计算抑瘤率;用免疫组织化学方法检测实验组和对照组移植瘤中CyclinD1和Caspase-3的表达水平。结果:黄芩苷组100mg/kg对裸鼠肝癌SMMC-7721实体瘤的抑制率为30.8%,两组间肝脏与肾脏指数比较,P均大于0.05。黄芩苷处理组凋亡指数为(13.6±1.1)%,高于对照组的(2.2±1.9)%,(P<0.01)。黄芩苷能够抑制CyclinD1表达及上调Caspase-3表达,(均P<0.05)。结论:黄芩苷对人肝癌SMMC-7721的裸鼠皮下移植瘤具有生长抑制作用,其抑瘤作用可能通过下调CyclinD1表达和上调Caspase-3表达来达到抑制瘤体增殖并有促进其凋亡的作用。在保护免疫器官功能方面,黄芩苷无明显优势。黄芩苷可能成为治疗原发性肝癌的有效手段之一,但是其在保护免疫器官方面还需要进一步研究。

消癌平与奥沙利铂及恩度联合对人肝癌细胞株SMMC-7721的体外增殖抑制实验

目的:通过体外实验研究消癌平(Xiaoaiping,XAP)注射液、奥沙利铂(oxaliplatin,L-OHP)、恩度(Endostar,重组人血管内皮抑制素)单药及其与XAP的两药及三药联合应用对人肝癌SMMC-7721增殖的影响,探讨其联合用药的理论依据.方法:采用MTT法观察XAP、L-OHP、恩度单药及其XAP的两药及三药联合应用对人肝癌SMMC-7721细胞增殖抑制作用,并观察细胞形态的变化.结果:10-40μL/mL XAP和1-4μg/mL L-OHP对SMMC-7721均有明显的抑制作用,并呈时间和浓度依赖性(P<0.01和P<0.05).1-12μg/mL恩度对该细胞无增殖抑制作用.20μL/mL XAP和1μg/mL L-OHP联合对SMMC-7721细胞增殖抑制率分别为82.08%,优于对照组(P<0.01),联合具有相加作用.20μL/mL XAP与6μg/mL恩度联合则具有明显的协同作用.三药联合抑制作用更明显.结论:XAP、L-OHP单药能抑制肝癌细胞增殖;恩度无抑制作用;与XAP两药联合对肝癌细胞增殖有协同增效作用,三药联合作用更明显.

环六亚甲基双乙酰胺诱导人肝癌SMMC-7721细胞分化过程中核基质-中间纤维系统的构型变化

目的:研究环六亚甲基双乙酰胺(HMBA)诱导人肝癌SMMC-7721细胞分化过程中细胞核基质-中间纤维系统的构型变化。方法:应用选择性抽提整装光镜与电镜技术观察SMMC-7721细胞诱导前后其核基质-中间纤维系统的构型特征。结果:对照组SMMC-7721细胞中,核基质纤维与中间纤维具有数量较少、单丝纤维较少、分布不均匀、排列无序、核纤层较厚、与两类纤维联系不紧密等特点,其核区域内可见一至数个由少量纤维维系着的残余核仁;而在经HMBA诱导处理后的分化细胞中,两类纤维不仅数量增多、层次丰富、分布更为均匀、单丝成份增多,而且核纤层更加致密,呈现薄层均一结构并与两类纤维发生更为紧密的联系,形成较为规则的纤维网架体系,其残余核仁由更加丰富且呈放射状排列的纤维所维系。结论:HMBA诱导体外培养人肝癌SMMC-7721细胞分化过程中的核基质-中间纤维系统的构型发生了明显的变化,这种变化是癌细胞恶性表型逆转的重要形态和功能表现。