The Effects of Serum from Patients with Acute Liver Failure on the Growth and Metabolism of Hep G2 Cells

In many bioartificial liver systems currently being designed and evaluated for use in fulminant hepatic failure, direct contact is required between the patient's blood and the liver cells in the device. The efficacy of such devices will be influenced by the interaction of fulminant hepatic failure (FHF) patient serum with the cells. We have found that FHF serum inhibits the growth rate and the synthesis of DNA, RNA, and protein; disturbs glutathione homeostasis; and induces morphological changes in cultured human Hep G2 cells. These interactions should influence the design of bioartificial liver devices based on proliferating cell lines and indicate the requirement to pretreat FHF patient plasma to reduce the toxin load.     

多途径丝裂原激活的蛋白激酶介导一氧化氮引起的肝癌细胞凋亡

目的研究非离子型的diazeniumdiolate类一氧化氮供体引起肝癌细胞凋亡的分子机制.方法利用免疫印迹、免疫沉淀、凝胶阻滞实验研究一氧化氮供体处理Hep3B肝癌细胞后,丝裂原激活的蛋白激酶、AP-1的激活以及和Hep3B肝癌细胞凋亡的关系.结果一氧化氮可引起细胞外信号调节蛋白激酶、c-jun N末端激酶和p38激酶的激活,特别是细胞外信号调节的蛋白激酶的持续激活,其中细胞外信号调节的蛋白激酶和c-jun N末端激酶的特异的阻断剂U0126和JNK抑制剂Ⅱ可阻断AP-1的激活和Hep3B细胞的凋亡,而p38激酶的阻断剂SB203580不能阻断AP-1的激活和Hep3B肝癌细胞的凋亡.结论一氧化氮通过激活细胞外信号调节蛋白激酶、c-jun N末端激酶,进而激活AP-1而引起Hep3B肝癌细胞的凋亡.     

有机污染物对人肝肿瘤细胞的遗传毒性

目的 用人肝肿瘤细胞HepG2／体外微核试验检测3种持久性有机污染物（POPs）Aroclor1254、毒杀芬和滴滴涕的遗传毒性。方法 人肝肿瘤细胞HepG2经Aroclor1254、毒杀芬和滴滴涕染毒24h，继续在补充细胞松弛素B（3μg/ml）的培养液中培养24h后，计数1000个双核细胞中的微核。结果 20，40μmol／L毒杀芬处理HepG2细胞的微核率与溶剂对照相比显著增加（P〈0.01，P〈0．01）；经Aroclor1254（23～184μmol／L）和滴滴涕（17．8～60μmol／L）处理的HepG2细胞的微核率与溶剂对照相比，差异无统计学意义。结论 Arodor1254和滴滴涕未显示对HepG2细胞明显的遗传毒性；毒杀芬可诱导HepG2细胞遗传损伤，有必要进一步评价其对人类健康的潜在危害。     

Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B,and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes

Human hepatic cell lines are widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins in the cell lines, including drug-metabolizing enzymes (DMEs), can differ significantly from those in human livers. In the present study, we first conducted an untargeted proteomics analysis of the microsomes of the cell lines HepG2, Hep3B, and Huh7, and compared them to human livers using a sequential window acquisition of all theoretical mass spectra (SWATH) method. Furthermore, high-resolution multiple reaction monitoring (MRM-HR), a targeted proteomic approach, was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines. In general, the SWATH quantifications were in good agreement with the MRM-HR analysis. Over 3000 protein groups were quantified in the cells and human livers, and the proteome profiles of human livers significantly differed from the cell lines. Among the 101 DMEs quantified with MRM-HR, most were expressed at substantially lower levels in the cell lines. Thus, appropriate caution must be exercised when using these cell lines for the study of hepatic drug metabolism and toxicity.     

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long‐term diseases including hepatocellular carcinoma (HCC ). Melatonin has been reported to alleviate promotion and progression of HCC , but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN ) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg^?1d^?1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK 1ε, Rev‐erbα, and Rev‐erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT 1 receptor and reduced levels of the hypoxia‐inducible factors Hif‐1α and Hif‐2α. An increased expression of p21, p53, and PARP 1/2, a higher Bax/Bcl‐2 ratio, and a lower expression of Cyclin D1, CDK 6, HSP 70, HSP 90, and GRP 78 proteins were also observed in melatonin‐treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV ‐ERB agonist SR 9009 in human Hep3B cells, and BMAL 1 knocking down attenuated the pro‐apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.     

Short-term Treatment of Daumone Improves Hepatic Inflammation in Aged Mice

Chronic inflammation has been proposed as one of the main molecular mechanisms of aging and age-related diseases. Although evidence in humans is limited, short-term calorie restriction (CR) has been shown to have anti-inflammatory effects in aged experimental animals. We reported on the long-term treatment of daumone, a synthetic pheromone secreted by Caenorhabditis elegans in an energy deficient environment, extends the life-span and attenuates liver injury in aged mice. The present study examined whether late onset short-term treatment of daumone exerts anti-inflammatory effects in the livers of aged mice. Daumone was administered orally at doses of 2 or 20 mg/kg/day for 5 weeks to 24-month-old male C57BL/6J mice. Increased liver macrophage infiltration and gene expression of proinflammatory cytokines in aged mice were significantly attenuated by daumone treatment, suggesting that short-term oral administration of daumone may have hepatoprotective effects. Daumone also dose-dependently suppressed tumor necrosis factor-α (TNF-α)-induced nuclear factor-κB (NF-κB) phosphorylation in HepG2 cells. The present data demonstrated that short-term treatment of daumone has anti-inflammatory effects in aged mouse livers possibly through suppression of NF-κB signaling and suggest that daumone may become a lead compound targeting aging and age-associated diseases.     

Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

Aim:

Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms.

Methods:

Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy.

Results:

Treatment of SMMC-7721 cells with Nutlin-3 (10 μmol/L) or etoposide (40 μmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells.

Conclusion:

Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.     

Metabolic Patterns (NAD(P)H) in Rat Basophilic Leukemia (RBL-2H3) Cells and Human Hepatocellular Carcinoma (Hep G2) Cells with Autofluorescence Imaging

Although the spatial and temporal distributions of cellular NAD(P)H concentrations have been theoretically predicted as typical patterns of the metabolism in living cells, so far such a pattern was observed only in neutrophils. In this work, the dynamic NAD(P)H distributions in rat basophilic leukemia (RBL-2H3) and human hepatocellular carcinoma (Hep G2) cells were studied by imaging the autofluorescence of cellular NAD(P)H with a sensitive CCD detector in a confocal microscope. The typical pattern of the cytoplasmic NAD(P)H wave traveling along the long axis of the elongated cell with a velocity of 2.2±0.6?μm/s was detected in RBL-2H3 cells. While in the case of Hep G2 cells, only the oscillation of the mitochondrial NAD(P)H was observed because the NAD(P)H mainly localized in mitochondria of Hep G2 cells. These results confirm the metabolic pattern of NAD(P)H in living cells and suggest that the expression of the metabolic pattern probably differs in different cell lines.     

siRNA干扰沉默S100A4基因表达对喉癌Hep2细胞周期和侵袭的影响

目的应用RNA干扰技术下调喉癌Hep2细胞中S100A4基因的表达,探讨其对Hep2细胞周期和细胞侵袭力的影响。方法脂质体法转染S100A4siRNA至Hep2细胞。Real-time PCR和Western blot验证S100A4基因mRNA和蛋白水平的表达,流式细胞术和transwell实验分别检测下调S100A4表达对Hep2细胞周期和侵袭能力的影响。结果 S100A4 mRNA和蛋白表达水平明显降低,细胞增殖指数显著降低,并且处于G1期细胞数明显增多,细胞侵袭能力显著降低。结论 S100A4siRNA转染喉癌Hep2细胞能有效下调S100A4基因的表达,从而影响Hep2的细胞周期和抑制细胞的侵袭能力。