Daintain/AIF-1强化了HepG2细胞顺铂耐药性的产生

目的：探讨炎症因子Daintain/AIF-1对肝癌细胞耐药性产生的影响。方法：利用MTT法测定耐药HepG2细胞株的IC50,流式细胞术测定耐药细胞株期凋亡率,HPLC方法检测隔耐药细胞株胞内顺铂的外排。结果：Daintain/AIF-1提高了HepG2耐药细胞株的IC50;再次受到相同剂量顺铂的攻击时,Daintain/AIF-1与顺铂联合运用构建的耐药细胞株凋亡率明显下降;Daintain/AIF-1促进了耐药细胞株胞内顺铂的外排。结论：此研究表明Daintain/AIF-1通过影响胞内顺铂的外排而促进了肝癌细胞对顺铂耐药性的产生。     

Daintain/AIF-1:一个疾病相关的多功能炎症因子

Daintain/AIF-1(大炎肽/同种异体移植炎症因子-1)是一个分子量17 kD的激素样活性蛋白,进化上具有保守性,内舍一个Ca2+结合的EF-手形结构域、一些PDZ结构域结合基序、以及一些其它的生物学活性位点.Daintain/AIF-1主要由巨噬细胞和激活的T淋巴细胞表达和分泌,是巨噬细胞/小胶质细胞激活和作用过程中的一个免疫调节器,并能调节血管平滑肌细胞和内皮细胞的增殖和迁移.Daintain/AIF-1作为一个与炎症相关的细胞因子,具有广泛的生物学特性,在机体的血管病变、移植排斥、炎症病变和自身免疫疾病以及癌症中发挥重要作用.     

青藏高原黑果枸杞花青素对人肝癌细胞增殖和自噬的影响

探究黑果枸杞花青素在体外对人肝癌HepG2细胞增殖和自噬的影响。利用CCK-8法测定细胞活力,EdU和细胞划痕试验检测细胞增殖和迁移效果,RT-PCR和Western blot检测增殖和自噬相关基因的mRNA和蛋白表达。结果显示,黑果枸杞花青素可有效抑制人肝癌HepG2细胞的增殖和迁移;上调增殖因子(LATS1、LATS2和MOB1)和自噬因子(Beclin-1、LC3-Ⅱ和AMPK),并下调增殖因子YAP的mRNA水平;下调自噬因子p-mTOR和细胞周期因子CDK4,并上调自噬因子p-AMPK和LC3-Ⅱ的蛋白表达。由此推测黑果枸杞花青素可在体外抑制人肝癌HepG2细胞增殖和迁移,并促进人肝癌HepG2细胞发生自噬。     

Daintain/AIF-1降低乳腺癌细胞MCF-7细胞的粘附能力

目的:探讨Daintain/AIF-1对乳腺癌细胞MCF-7粘附能力调控的机制。方法:制备Daintain/AIF-1条件培养基培养MCF-7细胞,分析细胞分泌成分的变化,特异性条带进行质谱分析,并通过RT-PCR和蛋白印迹进一步证实。结果:我们发现较高水平Daintain/AIF-1条件培养基培养后,可以上调MCF-7细胞中14-3-3ξ的表达水平,降低了细胞粘附能力。结论:本研究表明Daintain/AIF-1和14-3-3ξ这两种脚手架蛋白,可能通过影响细胞骨架以及细胞形态,从而影响乳腺癌的发展进程。     

小鼠Daintain/AIF-1基因启动子的克隆及其荧光素酶报告基因载体的构建

目的：对Daintain/AIF-1（大炎肽/同种异体移植炎症因子-1）基因启动子进行克隆并构建荧光素酶报告基因载体,为进一步研究Daintain/AIF-1的转录调控作用提供了质粒资源。方法：提取单核巨噬细胞系RAW264.7基因组DNA,以其为模板采用PCR方法克隆出Daintain/AIF-1基因5＇端UTR区1.6 kb DNA序列,将该序列同源重组到pGL3-Basic载体上,转化感受态DH5α并酶切鉴定和测序。结果：PCR产物片段与预期结果一致,Daintain/AIF-1基因5＇端UTR区1.6 kb DNA序列连接到pGL3-Basic载体上,构建成pGL3-Basic-Daintain/AIF-1（pGL3-Basic-DT）载体,酶切结果与理论预测值一致,经测序证实无碱基突变。结论：Daintain/AIF-1基因报告基因载体的构建为进一步研究Daintain/AIF-1转录调控作用提供了载体资源。     

白藜芦醇体外对肝癌HepG2细胞分化及P21^WAF1/CIP1表达的影响

目的：探讨白藜芦醇（Resvratrol,Res）在体外对肝癌细胞分化及相关周期蛋白依赖激酶抑制因子P21^WAF1/CIP1的影响。方法：体外培养肝癌HepG2细胞,用MTT法检白藜芦醇对HepG2细胞的生长抑制作用,用倒置显微镜观察肝癌细胞的形态改变,用放射免疫法检测其AFP分泌,以RT-PCR方法检测HepG2细胞中P21^WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21^WAF1/CIP1蛋白的表达。结果：白藜芦醇呈时间剂量性抑制HepG2细胞株的增殖,使其亚细胞结构趋于正常,AFP分泌量下降,并显著上调HepG2细胞中P21^WAF1/CIP1mRNA和蛋白的表达。结论：白藜芦醇能诱导HepG2细胞在体外向正常肝细胞分化,并上调其P21^WAF1/CIP1的表达。     

雷氏大疣蛛毒液对人肝癌HepG2细胞p21基因表达的影响

本文研究了雷氏大疣蛛毒液对人肝癌细胞株HepG2增殖抑制作用及其分子机制。采用XTT法观察到雷氏大疣蛛毒液剂量依赖抑制HepG2细胞增殖;流式细胞仪检测发现,经过雷氏大疣蛛毒液作用的HepG2细胞周期发生明显的选择性改变;RT-PCR方法检测到p21基因表达增强;Western blot检测发现,p21蛋白表达增加。结果提示,雷氏大疣蛛毒液抑制人肝癌细胞HepG2增殖的可能机制之一是使p21基因和蛋白表达增加,G2IM细胞周期被阻滞,从而诱导细胞凋亡。     

姜黄素对肝癌细胞HepG2的抗癌作用及P21WAF1/CIP1表达的影响

目的：探讨姜黄素对肝癌HepG2细胞抗癌作用及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1表达的影响。方法：体外培养肝癌HepG2细胞,用MTT法检测姜黄素对HepG2细胞的抑制作用,以RT-PCR方法检测HepG2细胞中P21WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1/CIP1蛋白的表达。结果：姜黄素呈时间剂量性抑制HepG2细胞的生长,并显著上调HepG2细胞中P21WAF1/CIP1mRNA和蛋白的表达。结论：姜黄素能抑制HepG2细胞的生长,并上调其中P21WAF1/CIP1的表达。     

小鼠Daintain/AIF-1 基因启动子的克隆及其荧光素酶报告基因 载体的构建

目的:对Daintain/AIF-1(大炎肽/同种异体移植炎症因子-1)基因启动子进行克隆并构建荧光素酶报告基因载体,为进一步研究Daintain/AIF-1的转录调控作用提供了质粒资源。方法:提取单核巨噬细胞系RAW264.7基因组DNA,以其为模板采用PCR方法克隆出Daintain/AIF-1基因5’端UTR区1.6 kb DNA序列,将该序列同源重组到pGL3-Basic载体上,转化感受态DH5α并酶切鉴定和测序。结果:PCR产物片段与预期结果一致,Daintain/AIF-1基因5’端UTR区1.6 kb DNA序列连接到pGL3-Basic载体上,构建成pGL3-Basic-Daintain/AIF-1(pGL3-Basic-DT)载体,酶切结果与理论预测值一致,经测序证实无碱基突变。结论:Daintain/AIF-1基因报告基因载体的构建为进一步研究Daintain/AIF-1转录调控作用提供了载体资源。     

白藜芦醇体外对肝癌HepG2细胞分化及P21WAF1/CIP1表达的影响

目的:探讨白藜芦醇(Resvratrol,Res)在体外对肝癌细胞分化及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1的影响.方法:体外培养肝癌HepG2细胞.用MTT法检白藜芦醇对HepG2细胞的生长抑制作用,用倒置显微镜观察肝癌细胞的形态改变,用放射免疫法检测其AFP分泌.以RT-PCR方法检测HepG2细胞中P21WAF1、CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1、CIP1蛋白的表迭.结果:白藜芦醇呈时间剂量性抑制HepG2细胞株的增殖,使其亚细胞结构趋于正常,AFP分泌量下降,并显著上调HepG2细胞中P21WAF1/CIP1 mRNA和蛋白的表达.结论:白藜芦醇能诱导HepG2细胞在体外向正常肝细胞分化,并上调其P21WAF1/CIP1的表达.     

Expression, purification, and characterization of functional recombinant human daintain/AIF-1 in Escherichia coli

Daintain/AIF-1 was identified from injured rat carotid arteries and porcine intestine in the mid 1990s. It is involved in autoimmune disorders, chronic rejection of allografts, gliomas, and breast cancer. Since it is convenient and economical to obtain such a peptide biologically, in this study, we describe the expression, purification, and characterization of recombinant human daintain/AIF-1 (rhdaintain/AIF-1). The backbone of vector pET32a, a high-level expression plasmid, was used to construct the pET32a-daintain/AIF-1 plasmid for daintain/AIF-1 expression in Escherichia coli. The recombinant daintain/AIF-1 protein was solubly expressed in the BL21 (DE3) strain and was purified by Ni2+ affinity chromatography. After purification, the recombinant protein showed the expected size of 18 kDa on Tricine-SDS-PAGE gels which was further confirmed by Western blotting. A total of 34.0 mg of high purity (over 98%) rhdaintain/AIF-1 was obtained from 1 L culture. The recombinant peptide was able to increase blood glucose elimination rates and enhance the proliferation of human MCF-7 cells. These results suggest that biological activity of the recombinant peptide was preserved after purification.     

Branched‐chain amino acids prevent insulin‐induced hepatic tumor cell proliferation by inducing apoptosis through mTORC1 and mTORC2‐dependent mechanisms

Branched‐chain amino acids (BCAA) supplementation has been reported to suppress the incidence of liver cancer in obese patients with liver cirrhosis or in obese and diabetic model animals of carcinogenesis. Whether BCAA directly suppresses cell proliferation of hepatic tumor cells under hyperinsulinemic condition remain to be defined. The aim of this study was to investigate the effects of BCAA on insulin‐induced proliferation of hepatic tumor cells and determine the underlying mechanisms. BCAA suppressed insulin‐induced cell proliferation of H4IIE, HepG2 cells. In H4IIE cells, BCAA did not affect cell cycle progression but increased apoptosis by suppressing expressions of anti‐apoptotic genes and inducing pro‐apoptotic gene via inactivation of PI3K/Akt and NF‐κB signaling pathways. Further studies demonstrated that BCAA inhibited PI3K/Akt pathway not only by promoting negative feedback loop from mammalian target of rapamycin complex 1 (mTORC1)/S6K1 to PI3K/Akt pathway, but also by suppressing mTORC2 kinase activity toward Akt. Our findings suggest that BCAA supplementation may be useful to suppress liver cancer progression by inhibiting insulin‐induced PI3K/Akt and subsequent anti‐apoptotic pathway, indicating the importance of BCAA supplementation to the obese patients with advanced liver disease. J. Cell. Physiol. 227: 2097–2105, 2012. © 2011 Wiley Periodicals, Inc.     

N-Benzoyl-12-nitrodehydroabietylamine-7-one, a novel dehydroabietylamine derivative, induces apoptosis and inhibits proliferation in HepG2 cells

The high biological activity of dehydroabietylamine derivatives has been reported previously. In this study, we aimed to screen 73 dehydroabietylamine derivatives as potential candidate inhibitors in liver cancer cells. Initially, the compounds structural activity relationship analysis was explored and N-benzoyl-12-nitrodehydroabietylamine-7-one (compound 81) was shown to have significant growth inhibitory activity in the human liver carcinoma cell line, HepG2. Further research into the anti-proliferative effect on HepG2 cells mediated by compound 81 was undertaken. The results suggest that compound 81 effectively induced apoptosis in HepG2 cells characterized by nuclear staining of DAPI, TUNEL assay and the activation of caspase-3. A decreased level of anti-apoptotic protein Bcl-2 and increased apoptotic Bax were also observed. Furthermore, Ki-67 protein staining and the BrdU incorporation assay showed that compound 81 significantly inhibited the proliferation of HepG2 cells. Cell cycle components analysis found that expression of cyclin D1 and cyclin B1 was reduced in HepG2 cells with compound 81 treatment, whereas the content of p21(Waf1/Cip1) was increased. Taken together, our data indicate that compound 81 induces apoptosis and inhibits proliferation in HepG2 cells, and may be a promising candidate in the development of a novel class of antitumor agents.     

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

Genistein is an isoflavonic phyto-oestrogen contained in soya beans. It is thought to display anti-cancer effects. This study was designed to investigate its effect on human intestinal colon cancer Caco-2 cells. MTT assay, flow cytometric analysis and western blotting were used to investigate the effect of genistein on cell proliferation, cell cycle progression and protein alterations of selected cell cycle-related proteins in Caco-2 cells. Our results showed that genistein and daidzein significantly suppressed cell proliferation. Genistein treatment was demonstrated to modulate cell cycle distribution through accumulation of cells at G2/M phase, with a significant decreasing effect of Cyclin B1 and Serine/threonine-protein kinase 2 (Chk2) proteins expression. However, daidzein did not alter the cell cycle progression in Caco-2 cells. All these observation strongly indicate that genistein has anti-proliferative effect in human intestinal colon cancer Caco-2 cells through the down-regulation of cell cycle check point proteins, Cyclin B1 and Chk2.     

珊瑚树Vibsane型二萜类化合物对人体HepG2细胞增殖的影响及其机制

目的：探讨珊瑚树vibsane型二萜类化合物对肝癌HepG2细胞增殖的影响及其机制,为研发新型天然植物类抗肿瘤药物提供实验依据。方法：采用噻唑蓝比色法及苔盼蓝染色计数法观察珊瑚树vibsane类二萜类化合物对不同肿瘤细胞增殖的影响;应用流式细胞仪检测细胞周期及细胞凋亡,利用Apo-ONE Homogeneous Caspase-3/7试剂盒检测vibsane二萜类化合物1#对HepG2细胞内Caspase-3酶活性的影响。结果：活性筛选发现vibsane型二萜类化合物1#显著抑制人肝癌HepG2细胞增殖,构效分析表明化合物C11位连接侧链的基团修饰影响其细胞增殖抑制活性。此外,HepG2细胞对1#化合物最敏感,1#化合物抑制其增殖具有剂量和时间依赖性。机制研究显示1#化合物诱导HepG2细胞发生明显的细胞周期G0/G1期阻滞,具有时间和剂量效应;同时,较高浓度1#化合物（5-10μmol/L）引起HepG2细胞凋亡明显增加,并剂量依赖性诱导细胞内Caspase3/7激活。结论：珊瑚树vibsane型二萜类化合物能够明显抑制人肝癌HepG2细胞增殖,其可能通过诱导细胞周期阻滞和细胞凋亡发挥抗肿瘤作用。     

Delisheng,a Chinese medicinal compound,exerts anti-proliferative and pro-apoptotic effects on HepG2 cells through extrinsic and intrinsic pathways

The anti-proliferative, cytotoxic and apoptogenic activities of delisheng, a Chinese medicinal compound, has been investigated. In this study, the hepatocarcinoma cell line (HepG2) and the liver cell line (L-02) were exposed to delisheng (6.25, 50 and 100 μl/ml). Delisheng suppressed the proliferation and viability of normal liver L-02 cells slightly, but strongly inhibited the proliferation and viability of hepatocarcinoma HepG2 cells. The flow cytometric analysis of HepG2 cells demonstrated that delisheng primarily arrested the HepG2 cells at the G1 phase of the cell cycle. Annexin V-FITC/PI staining corroborates the apoptogenic nature of delisheng on HepG2 cells. The anti-proliferative and pro-apoptotic effect of delisheng in HepG2 cells was associated with changes in the Bcl-2/Bax ratio and the induction of caspase-mediated apoptosis. Upregulation of DR5 expression was observed in HepG2 cells after treatment with delisheng. The findings from the present study suggest that delisheng has selective cytotoxic activities against HepG2 cells. Delisheng triggered time- and dose-dependent apoptosis in HepG2 cells by activating the mitochondria-mediated and death receptor-mediated apoptotic pathways.     

顺铂对HepG2细胞增殖、细胞周期及肝癌干细胞标志物的影响

目的:研究顺铂对HepG2细胞增、细胞周期及肝癌干细胞标志物(CD133、ICAM-1和ABCG2)的影响。方法:选取HepG2作为研究对象,分别采用MMT比色法、PI染色法及免疫荧光法检测不同浓度顺铂对其增殖、细胞周期、CD133、ICAM-1和ABCG2表达的影响。结果:每个浓度顺铂作用后均可以显著抑制HepG2细胞增殖力(F=12.23,P=0.004);顺铂对HepG2细胞增殖力的抑制作用和浓度可能与时间成正比。0 mg/L组静息期(G0/G1期)细胞比例为(50.25±0.79)%、2 mg/L组G0/G1期细胞比例为(89.24±0.41)%、4 mg/L组G0/G1期细胞比例为(29.54±3.02)%,2 mg/L组和4 mg/L组分别比0 mg/L组显著上升和下降,差异明显有统计学意义(t=6.53、-3.65,均P0.05)。0 mg/L组DNA合成期(S期)细胞比例为(47.13±0.74)%、2 mg/L组S期细胞比例为(5.65±0.42)%、4 mg/L组S期细胞比例为(67.46±3.24)%,2 mg/L组和4 mg/L组分别比0 mg/L组显著下降和上升,差异明显有统计学意义(t=-7.35、3.79,均P0.05)。结果提示2 mg/L组和4 mg/L组顺铂可让HepG2在G0/G1期与S期显著阻滞,差异有统计学意义(P0.01);顺铂处理后,剩余的HepG2细胞的CD133、ICAM-1和ABCG2呈现高表达水平。结论:HepG2细胞系中会有很少部分肝癌干细胞避开了顺铂的杀灭作用,是造成临床上肝癌反复发作的重要因素之一,临床上应予以重视。