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 proteinⅠ, 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.     

Alteration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

AIM: To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS: Cells cultured with or without 5 x 10(-3) mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www.matrixscience.com). RESULTS: The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched ultrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pyst1, hypothetical protein, nucleophosmin 1, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, beta-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION: The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament (NM-IF) system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.     

重组腺病毒介导的人AWP1基因转移对人源肝癌细胞增殖的影响

目的以重组腺病毒作为AWP1（associated with PRK1）的转基因载体,研究AWP1对人肝癌细胞增殖的影响,为进一步研究AWP1基因功能提供依据。方法重组腺病毒质粒pAd-flag-AWP1转染293细胞进行重组腺病毒的包装和扩增,并进行PCR鉴定。用空病毒Ad-Null和重组腺病毒Ad-flag-AWP1分别感染人源肝癌细胞SMMC-7721,并于感染后24 h、72 h、第5天和第7天收集细胞计数,绘制细胞生长曲线,计算细胞倍增时间。结果重组腺病毒Ad-flag-AWP1能够感染SMMC-7721细胞;依据细胞生长曲线计算的SMMC-7721细胞倍增时间分别为：SMMC-7721细胞约为5 d;而Ad-Null感染后其生长被抑制,倍增时间约为6 d;Ad-flag-AWP1感染后,其倍增时间约为3 d。结论重组flag-AWP1腺病毒转染SMMC-7721细胞过表达的AWP1蛋白可能具有促进人肝癌细胞生长的作用。     

3-溴丙酮酸对SMMC-7721肝癌细胞增殖的抑制作用

目的观察3-溴丙酮酸（3-BrPA）对SMMC-7721肝癌细胞增殖的抑制作用。方法对经0．25mmol／L～8．00mmol／L3-BrPA溶液处理的SMMC-7721细胞,采用MTT法检测细胞增殖抑制情况,并在荧光显微镜和电镜下观察细胞内部结构的变化。结果3-BrPA对SMMC-7721细胞的增殖有明显的抑制作用,其中在0．75mmol／L浓度下的抑制作用最强;经处理后的细胞,在荧光染色下显示坏死细胞增多,在电镜下显示细胞染色质分散,少量核染色质凝聚。结论3-BrPA对SMMC-7721肝癌细胞有抑制作用。     

新城疫病毒HN基因诱导肝癌细胞SMMC7721凋亡的作用机制

目的 探索含新城疫病毒(NDV)HN基因的质粒pVHN诱导人肝癌细胞SMMC7721凋亡的作用及其机制。方法 以脂质体介导方法在体外转染pVHN于SMMC7721细胞24h后,通过MTT方法检测细胞活性状态;采用流式细胞仪(FCM)检测法,通过碘化丙啶(PI)染色测定细胞死亡率;以罗丹明123染色测定线粒体跨膜电位(△ψ)的改变;提取细胞基因组DNA进行电泳,检测DNA有无断裂;用底物颜色反应检测Caspase-3活性。结果 细胞SMMC7721在体外转染pVHN后,死亡率达50．0％(转染空载体质粒对照组为5．2％);细胞基因组DNA呈梯状谱带;线粒体△ψ下降,Caspase-3活性明显升高。结论 新城疫病毒HN基因在体外转染细胞SMMC7721,能明显诱导细胞SMMC7721凋亡,其发生机制可能是由于HN基因的导入引起线粒体△ψ下降,进而激活Caspase-3使细胞凋亡。     

Expression of TN4 gene and its role in human hepatocarcinogenesis from Qidong, a liver cancer risk area

Background We investigated the expression and role of TN4 in the oncogenesis of human hepatocellular carcinoma (HCC) from Qidong which is a HCC risk area.Methods The expression of TN4 in HCC was observed using immunohistochemical staining (IHC). TN4 levels were manipulated in human liver cancer cell SMMC7721, using pcDNA3.1 eukaryotic expression constructs designed to express the complete TN4 cDNA. The biological changes of the cells were observed before and after transfection of TN4 and the change of gene expression was analysed by atlas cDNA expression array. Results Among 100 pairs of samples of HCC, TN4 down-regulation expression and up-regulation expression positive rate were 81% (81/100), 19% (19/100), respectively (P<0.01). TN4 protein was mainly localized in cytoplasm and membrane. The positive rate of TN4 were 10% (3/30), 100% (70/70) in lymph node metastasis and no lymph node metastasis, respectively (P<0.01).　The growth rates of the derivative SMMC7721-TN4 cell lines were decreased in comparasion with that of normal SMMC7721 cells and pcDNA- SMMC7721. Some gene expression was changed before and after transfection of TN4. At 30 days of post-implantation of SMMC7721-TN4, SMMC7721-pcDNA3, SMMC7721 group produced tumors of (301.9±143.4) mm3, (2418.7±362.8) mm3, (2317.4±587.8) mm3, respectively, (P<0.01). Tumor inhibiting rate was 82.4% in TN4 transfection group. Sections of tumors were observed for their degree of tissue necrosis and there was higher degree of necrosis in tumors of the TN4-SMMC7721 cell group than those of the SMMC7721, SMMC7721-pcDNA groups.Conclusions TN4 may play an important role in the oncogenesis of human HCC, especially in Qidong, the HCC risk area and TN4 could be a candidate tumor suppressor gene for HCC.     

血清药理学方法观察复方斑蝥胶囊对人肝癌细胞SMMC-7721增殖的影响

目的 :研究复方斑蝥胶囊含药动物血清对人肝癌细胞SMMC -7721增殖的影响。方法 :采用血清药理学方法 ,对兔灌胃给予不同剂量 (临床等效剂量、2倍临床等效剂量和3倍临床等效剂量 )的复方斑蝥胶囊混悬液 ,应用MTT比色法观察含药血清对人肝癌细胞SMMC -7721增殖的抑制作用 ,同时观察临床等效剂量组各采血时间点的抑制作用。结果 :3个剂量组的含药血清对SMMC -7721细胞增殖均有抑制作用 (P<0 01) ,其中以3倍临床等效剂量组含药血清的抑制率最高 ;临床等效剂量组各采血时间点的含药血清对SMMC -7721细胞增殖均有抑制作用 (P<0 05) ,其中3h时含药血清的抑制率最高。结论 :复方斑蝥胶囊含药动物血清对人肝癌细胞SMMC -7721增殖具有一定的抑制作用 ,并具有剂量依赖性。     

桔皮素对人肝癌细胞株SMMC-7721抑制作用的初步研究

目的：探讨桔皮素对人肝癌细胞株SMMC-7721的抑制作用。方法：采用四唑盐（MTT）比色法观察不同浓度桔皮素对人肝癌细胞株SMMC-7721细胞增殖的影响，计算半数抑制浓度IC50；绘制细胞株SMMC-7721的生长曲线观察桔皮素对其增殖的影响。结果：MTT结果显示不同浓度的桔皮素对SMMC-7721的生长均有一定的抑制作用，同一时间，各浓度组与对照组相比均有统计学差异（P〈0．05）；桔皮素浓度为0．24—30μg·mL。时，对SMMC-7721细胞在药物处理72小时后抑制率为3．85％～93．59％，且72小时的IG50为14．69—21．11μg·mL^-1。生长曲线结果提示，桔皮素对SMMC-7721细胞的抑制作用呈明显时效和量效关系。结论：桔皮素能抑制人肝癌细胞株SMMC-7721的增殖。     

不同中医治法对不同肝癌细胞株的作用比较

目的 :比较不同中医治法对不同肝癌细胞株增殖的抑制作用。方法 :将不同治法的中药常规水煎醇沉后 ,采用不同浓度中药制剂直接加入培养液的给药方式 ,以MTT法测定人原发性肝癌SMMC772 1细胞株、Bel74 0 2细胞株细胞的增殖情况。结果 :①清热解毒法对两种不同人肝癌细胞株增殖的抑制作用都很明显 ,且随用药剂量的增大 ,呈量效关系 ;②活血化瘀法对SMMC772 1细胞株增殖也有较好抑制作用 ,而对Bel74 0 2细胞株增殖却作用不明显 ;③健脾理气法对SMMC772 1细胞株增殖无抑制作用 ,而对Bel74 0 2细胞株增殖却有较好作用 ;④清热解毒法与其它两治法配伍后 ,其对SMMC772 1细胞株增殖的抑制作用都不如单一清热解毒法。结论 :清热解毒法对不同肝癌细胞株增殖具有较强的抑制作用 ,支持临床大剂量使用这类中药 ,活血化瘀法与健脾理气法具有选择性的抑制作用 ,说明临床辨证论治的重要性     

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。