Daintain/AIF-1促进肝癌细胞的增殖与迁移

目的：探讨炎症因子Daintain/AIF-1在肝癌发生发展进程中的作用。方法：利用结晶紫染色方法测定HepG2细胞的增殖,流式细胞术测定细胞周期分布,western blot方法检测相关周期表达蛋白,Transwell方法检测HepG2细胞的迁移。结果：在此研究中我们发现Daintain/AIF-1通过上调周期相关蛋白cyclinD1和cdk4的表达以及增加Rb的磷酸化,加快了HepG2细胞周期的进程,从而促进了HepG2细胞的增殖,另外我们发现Daintain/AIF-1也促进了HepG2细胞的迁移。结论：此研究表明Daintain/AIF-1参与了肝癌的发生发展进程,更进一步证明了炎症因子与癌症的发生发展密不可分。     

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47^phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ _m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ _m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47^phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47^phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47^phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.     

siRNA 诱导CyclinB1沉默增加HePG2细胞对柔红霉素的敏感性

探讨了CyclinB1在肝癌药物耐受形成中的作用.用siRNA技术沉默CyclinB1在细胞中的表达,使用流式细胞仪技术分析细胞的凋亡和周期分布,用细胞克隆形成能力分析和细胞毒性实验分析细胞的增殖能力.由siRNA诱导的CyclinB1下调导致40%～50%肝癌细胞阻滞在G2/M期,并显著抑制肝癌细胞的单克隆形成能力;柔红霉素联合CyclinB1 siRNA较单独使用柔红霉素能更加有效地导致肝癌细胞凋亡,而在人正常肝细胞HL-7702中这种现象不明显.实验结果表明针对于CyclinB1的靶向性下调与肝癌药物的联合使用将有可能成为肝癌治疗的新策略.     

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。     

mcl-1特异性siRNA对HepG2细胞增殖及凋亡的影响

目的：肝癌分子靶向治疗是目前研究的热点,肝癌相关基因mcl-1在肝癌增殖及凋亡中的作用尚不明确,本研究拟探讨mcl-1特异性siRNA对体外培养肝癌细胞HepG2增殖及凋亡的影响。方法：设计、合成有效的mcl-1特异性siRNA序列,体外转染HepG2细胞;通过绘制细胞生长曲线和MTT实验检测mcl-1特异性siRNA对HepG2细胞增殖的影响;通过AnnexinV／PI双标记流式细胞仪检测mcl-1特异性siRNA对HepG2细胞凋亡率的影响。结果：通过绘制细胞生长曲线发现,mcl-1特异性siRNA能够抑制HepG2细胞的增殖（P〈0．05）,MTT实验提示转染mcl-1特异性siRNA24h、48h、72h后,HepG2细胞存活率均显著下降（P〈O．05）;流式细胞仪检测分析发现,转染mcl．1特异性siRNA后AnnexinV＋／PI-细胞百分率显著增高（P〈0．01）,提示mcl-1具有促进HepG2细胞凋亡的作用。结论：Mcl-1蛋白具有促进肝癌细胞增殖,抑制肝癌细胞凋亡的作用,这种分子特性符合肿瘤靶向治疗的要求,Mcl-1可能成为肝癌靶向治疗的潜在靶点。     

人参皂苷Rb1对油酸诱导HepG2细胞脂肪堆积的影响作用

通过建立体外肝细胞脂肪堆积模型评价人参皂苷Rb1清除肝细胞脂肪堆积的能力.方法:1 mmol· L-1油酸诱导建立HepG2细胞脂肪堆积模型,从噻唑兰染色吸光度(MTT值)、甘油三酯(TG值)、细胞内脂滴形态3方面评价人参皂苷Rb1的作用.结果:人参皂苷Rb1可明显减轻细胞内脂质堆积现象,显著降低细胞中TG含量,其中100 μg·mL-1人参皂苷Rb1对TG的清除率达37.9％.结论:人参皂苷Rb1具有良好的体外降脂活性,对脂肪肝有较好的预防效果.     

Evaluating the Extent of LINE-1 Mobility Following Exposure to Heavy Metals in HepG2 Cells

The long interspersed elements-1 (LINE1 or L1 retrotransposon) constitute 17 % of the human genome and retain mobility properties within the genome. At present, 80–100 human L1 elements are thought to be active in the genome. The mobilization of these active elements may be influenced upon exposure to the heavy metals. In the present study, we evaluated the association of aluminum, lead, and copper exposure with L1 retrotransposition in human hepatocellular carcinoma (HepG2) cell line. An in vitro retrotransposition assay using an enhanced green fluorescent protein (EGFP)-tagged L1RP cassette was established to track EGFP shining as the mark of retrotransposition. Following determination of noncytotoxic concentrations of these metals, pL1RP-EGFP-transfected HepG2 cells were subjected to long-term treatment. Flow cytometry analysis of cells treated with various concentrations of these metals along with quantitative real-time PCR was used to quantify L1 retrotransposition frequencies. Aluminum significantly increased L1 retrotransposition frequency, while no significant association was found concerning lead exposure and L1 retrotransposition. Copper treatment downregulated L1 retrotransposition as a result of EGFP-tagged L1RP expression. Our findings suggest that aluminum might have the potential to cause genomic instability by the enhancement of L1 mobilization. Thus, the risk of induced L1 retrotransposition should be considered during drug safety evaluation and risk assessments of exposure to toxic environmental agents. Further studies are needed for a more robust assay to evaluate any associations between long-term lead exposure and L1 mobility in cell culture assay.     

沉默FNBP1表达诱导7703细胞形态重塑

FNBP1在真核细胞中广泛表达,能通过诱导细胞膜管状化、内陷或变形及后续膜曲率依赖性肌动蛋白聚合过程,参与细胞内吞和运动;还可参与端粒维持及FasL与溶酶体结合过程的调控。研究发现,在肝癌细胞7703中,利用RNA干扰（RNA interference technology,RNAi）技术使内源FNBPI基因表达沉默以后,7703细胞形态发生重塑,由上皮样转变为树状分枝的纤维状,表明FNBP1在细胞形态维持过程中亦具有不可或缺的潜在作用;通过对已知FNBP1蛋白相互作用情况的分析推断,FNBP1可能是通过对肌动蛋白骨架动态组装过程的分子调控参与7703细胞形态的控制。     

NOR1基因转染对肝癌细胞HepG2基因表达谱的影响

NOR1基因是一在正常组织中广泛表达且在肿瘤组织中表达下调的新基因.为进一步研究NOR1基因的功能和寻找其下游基因,利用脂质体技术将NOR1基因转染进HepG2细胞,采用cDNA微阵列技术分析其基因表达谱的改变.试验表明NOR1基因的转染能使Grb2,HBP17,TNFRSF11B等59个基因上调,同时也下调Bik,MAp2K6,ZFP95等103个基因.随后用实时荧光定量PCR对cDNA 微阵列结果中上述3个上调表达基因进行验证,结果表明,基因表达差异具有统计学意义(P<0.05),荧光定量PCR结果与微阵列结果相符.这些结果提示,NOR1基因对肝癌HepG2细胞的生物学行为的影响可能与它对细胞信号转导,细胞周期调控,转录、翻译调控相关基因的表达影响有关.     

HCV 1a/1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a/1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Western blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV)1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达.结果证明,转染细胞中检测到分子量约70 kDa的HCV NS3蛋白, 转染细胞连续传20代, 仍能检测到HCV正、负链RNA.表明该HCV嵌合体可以在细胞中复制和表达,HCV 1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制. HCV 5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合.3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失"A",9409,9410位及9495,9496,9497位分别插入"TT"和"AAT"可不影响RdRp的生物活性.本研究对阐明HCV复制和翻译机制有重要意义.     

HCV 1a／1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a／1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Westem blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV) 1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达。结果证明,转染细胞中检测到分子量约70kDa的HCV NS3蛋白,转染细胞连续传20代,仍能检测到HCV正、负链RNA。表明该HCV嵌合体可以在细胞中复制和表达,HCV1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制。HCV5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合。3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失“A”,9409,9410位及9495,9496,9497位分别插入“TT”和“AAT”可不影响RdRp的生物活性。本研究对阐明HCV复制和翻译机制有重要意义。     

Lipid Efflux Mediated by Alkylphospholipids in HepG2 Cells

Antitumoural alkylphospholipid (APL) analogues alter cholesterol homoeostasis in HepG2 cells by interfering with cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and at the same time stimulating the release of considerable quantities of membrane cholesterol. The capacity of APLs to stimulate cholesterol efflux is suppressed when cells are incubated simultaneously with APLs and serum whilst the inhibition of cholesterol transport to the ER (measured in terms of the synthesis of esterified cholesterol) persists, indicating that both effects are independent of each other. Interestingly, our results suggest that both raft and non-raft membrane domains contribute to the cholesterol released to APLs. In addition, a marked efflux of choline-bearing phospholipids (phosphatidylcholine (PC) and sphingomyelins (SM)) was found to be related to this release of cholesterol. Finally, we observed that APL micelles composed of cholesterol might act as donor/acceptor cholesterol systems. Thus, the findings of this study clearly demonstrate that antitumoural APLs act as extracellular acceptors, stimulating cholesterol and phospholipid efflux, although they may also play a role as cholesterol donors.     

5- 脱氧杂氮胞苷对人肝癌细胞HepG2 增殖及beclin1 表达的影响

目的:观察5-脱氧杂氮胞苷(5-aza-CdR)对HepG2细胞生长抑制及beclin1表达的影响,以探讨其抗肿瘤发生的潜在机制。方法:采用四甲基偶氮唑盐(MTT)法检测5-aza-CdR对HepG2细胞的生长抑制;用相差显微镜观察不同药物浓度下不同时间段的肝癌细胞形态学改变;采用RT-PCR法和Western blot法检测5-Aza-CdR对抑癌基因beclin1的mRNA和蛋白表达的影响。结果:5-aza-CdR可抑制HepG2细胞生长,呈剂量依赖性,并上调beclin1的mRNA和蛋白的表达。结果:显示102.4umol/L5-aza-C-dR作用72小时细胞增殖抑制率最高,可达(84.3±3.31)%,beclin1的mRNA和蛋白表达上调最明显,与对照组相比差异有统计学意义。结论:5-aza-CdR可抑制HepG2细胞增殖,其机制可能是通过恢复某些抑癌基因的表达,上调beclin1的mRNA和蛋白表达。     

Metabolic profile and quantification of deoxyribose synthesis pathways in HepG2 cells

Traditional tracer studies of cell proliferation fail to distinguish between label enrichment due to increased DNA repair versus DNA replication. We used the emerging stable (non-radiating) isotope-based dynamic metabolic profiling technique on HepG2 cells to determine synthesis pathways of nucleic acids from glucose and rates of proliferation using CG-MS assay of RNA and DNA enrichment. Comparing the isotopic enrichment curve in DNA with the theoretical curve based on cell growth, we observed that the measured tracer enrichment was significantly higher, indicating that surplus label was acquired during DNA repair. In particular, after the first duplication (3 days), 80.13% of the total enrichment observed corresponds to duplication and 19.87% corresponds to DNA repair as calculated from the [1, 2-¹³C₂]-glucose incorporation curve. Our data indicate contemporary measurements of cell proliferation rates relying on tracer incorporation may be overestimated. ¹³C label was distributed between m1 (m1/Σm = 80) and m2 (m2/Σm = 14) of deoxyribose, indicating that most of the glucose carbon was acquired via direct glucose oxidation in the pentose cycle. The stable isotope technique distinguishes rates of DNA synthesis and repair via the oxidative and non-oxidative pentose cycle, separately, in one test, without inhibition of either process. The contribution of DNA repair in malignant cells to isotope accumulation in deoxyribose remains to be investigated.     

根皮素对HepG2肝癌细胞增殖和凋亡影响的实验研究

目的：探讨根皮素对人肝癌HepG2细胞增殖和凋亡的影响。方法：MTT法检测不同浓度（30、40、50 ug/mL）根皮素对肝癌 HepG2 细胞增殖的抑制作用,流式细胞技术检测根皮素对HepG2细胞凋亡的影响,ELISA 法检测不同浓度根皮素干预后细胞中 Bcl-2 和Bax表达的变化,Western blot法检测30 ug/mL根皮素在8,16,24 小时后检测p-AKT 蛋白表达情况。结果：30、40 和50 ug/mL 的根皮素对肝癌HepG2 细胞的增殖均有抑制作用（P＜0.01）,同时,30、40 和50 ug/mL 的根皮素在24 小时后可诱导 HepG2 细胞发生早期凋亡,凋亡率分别为0.1321± 0.0224, 0.2607± 0.0457, 0.3712± 0.0884（P＜0.01）;另外,30、40 和50 ug/mL的 根皮素作用24 小时后细胞内Bcl-2 表达降低,Bax 表达增高（P＜0.01）;最后,30 ug/mL根皮素可以明显减少p-AKT 表达量,这 种作用呈现时间依赖性。结论：根皮素能够抑制肝癌HepG2 细胞的增殖和促进细胞凋亡。     

Cytochrome P450 1A1 (CYP1A1) Catalyzes Lipid Peroxidation of Oleic Acid-Induced HepG2 Cells

Nonalcoholic fatty liver disease (NAFLD) is a chronic hepatic disease associated with excessive accumulation of lipids in hepatocytes. As the disease progresses, oxidative stress plays a pivotal role in the development of hepatic lipid peroxidation. Cytochrome P450 1A1 (CYP1A1), a subtype of the cytochrome P450 family, has been shown to be a vital modulator in production of reactive oxygen species. However, the exact role of CYP1A1 in NAFLD is still unclear. The aim of this study was to investigate the effects of CYP1A1 on lipid peroxidation in oleic acid (OA)-treated human hepatoma cells (HepG2). We found that the expression of CYP1A1 is elevated in OA-stimulated HepG2 cells. The results of siRNA transfection analysis indicated that CYP1A1-siRNA inhibited the lipid peroxidation in OA-treated HepG2 cells. Additionally, compared with siRNA-transfected and benzo[a]pyrene (BaP)-OA-induced HepG2 cells, overexpression of CYP1A1 by BaP further accelerated the lipid peroxidation in OA-treated HepG2 cells. These observations reveal a regulatory role of CYP1A1 in liver lipid peroxidation and imply CYP1A1 as a potential therapeutic target.     

Activation of Human Stearoyl-Coenzyme A Desaturase 1 Contributes to the Lipogenic Effect of PXR in HepG2 Cells

The pregnane X receptor (PXR) was previously known as a xenobiotic receptor. Several recent studies suggested that PXR also played an important role in lipid homeostasis but the underlying mechanism remains to be clearly defined. In this study, we found that rifampicin, an agonist of human PXR, induced lipid accumulation in HepG2 cells. Lipid analysis showed the total cholesterol level increased. However, the free cholesterol and triglyceride levels were not changed. Treatment of HepG2 cells with rifampicin induced the expression of the free fatty acid transporter CD36 and ABCG1, as well as several lipogenic enzymes, including stearoyl-CoA desaturase-1 (SCD1), long chain free fatty acid elongase (FAE), and lecithin-cholesterol acyltransferase (LCAT), while the expression of acyl:cholesterol acetyltransferase(ACAT1) was not affected. Moreover, in PXR over-expressing HepG2 cells (HepG2-PXR), the SCD1 expression was significantly higher than in HepG2-Vector cells, even in the absence of rifampicin. Down-regulation of PXR by shRNA abolished the rifampicin-induced SCD1 gene expression in HepG2 cells. Promoter analysis showed that the human SCD1 gene promoter is activated by PXR and a novel DR-7 type PXR response element (PXRE) response element was located at -338 bp of the SCD1 gene promoter. Taken together, these results indicated that PXR activation promoted lipid synthesis in HepG2 cells and SCD1 is a novel PXR target gene.     

Metabolic Reprogramming in Mutant IDH1 Glioma Cells

BackgroundMutations in isocitrate dehydrogenase (IDH) 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS)–detectable changes in the cellular metabolome.MethodsTwo genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA)-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by ¹H-MRS. Principal Component Analysis was used to analyze the MRS data.ResultsPrincipal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.ConclusionThe IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.