Abstract: Lipid metabolism is an important section of human body metabolism, and lipid metabolism disorder can lead to multiple diseases. Canarium album is a nature food, whose extract has been reported hepatoprotective, anti-inflammatory and antioxidant. In this study, we extracted polyphenol and flavonoid substances from Canarium album fruits, and demonstrated that they restrain lipid excessive accumulation induced by oleic acid in hepatocarci- noma cells. Moreover, polyphenol and flavonoid extracted from Canarium album fruits facilitated phosphorylation of adenosine monophosphate activated protein kinase (AMPK) and regulated several lipid metabolism related genes expression, including fatty acid synthase (FAS), sterol regulatory element binding protein (SREBP)-1 and peroxisame proliferator activated receptor (PPAR)-α. Therefore, for the first time, we demonstrated that Canarium album extract restrained lipid excessive accumulation by activating AMPK signaling pathway, downregulating SREBP-1 and FAS, upregulating PPAR-α in hepatocarcinoma cells, which may be of great significance for prevention and clinical treat- ment of lipid metabolism disorders.
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This study mainly examines the expression differences of the SDF1 ligand and the CXCR4 receptor in mouse hepatocarcinoma cells with different lymphatic metasta- sis abilities and in mouse normal liver cells in vitro and in vivo. This study reveals the effects of AnnexinA7 downregulation and upregulation on the SDF1/CXCR4 axis in mouse liver cancer cells in vitro and in vivo. Ac- cording to previous reports and the results of our stud- ies, the mechanisms of SDF1/CXCR4 in tumor development and progression may be as follows: to stimulate tumor cell autocrine and paracrine growth sig- naling factors, provoking cell proliferation and anti-apoptosis; to cooperate with other molecular net- works, strengthening the activity of related pathways; to adjust the synthesis and degradation of actins and extracellular matrix, causing tumor cell movement and invasion; and to regulate the activity of adhesion molecules on tumor cell surfaces, enhancing the rec- ognition, chemotaxis and adhesion to endothelial cells. However, it remains unknown how the gener- ation and secretion of SDF1/CXCR4 molecules correl- ate in the same cancer cell and how they interact with upstream and downstream pathways, e.g., AnnexinA7 and VEGFC/D-VEGFR3/NRP2 during tumor development and progression. Thus, further studies are required.
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immunosuppression. To examine the role of cell cycle and cellular differentiation on liver cell susceptibility to reovirus infection, a murine hepatocarcinoma cell line, Hepa 1/A1, was infected with reovirus and assayed for the presence of infectious virus or reovirus antigen in cells. Despite a > 95% binding of reovirus to hepatocarcinoma cells as indicated by
Abstract: Alumina nanoparticles (Al 2 O 3 NPs) are gradually used in various areas, including nanomedicine, biosensors, and electronics. The current study aimed to explore the DNA damage and cytotoxicity due to Al 2 O 3 NPs on human hepatocarcinoma cells (HepG2). The MTT and neutral red uptake assays showed that Al 2 O 3 NPs induce significant cell death in a dose- and time-dependent manner. However, Al 2 O 3 NPs induced significant intracellular reactive oxygen species production and elevated lipid peroxidation and superoxide dismutase levels in the HepG2 cells. Al 2 O 3 NPs also induced significant decrease in reduced glutathione levels and increase caspase-3 activity in HepG2 cells. DNA fragmentation analysis using the alkaline single-cell gel electrophoresis showed that Al 2 O 3 NPs cause genotoxicity in dose- and time-dependent manner. However, they induce reactive oxygen species production and oxidative stress, leading to oxidative DNA damage, a probable mechanism of genotoxicity. This study warrants more careful assessment of Al 2 O 3 NPs before their industrial application.
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Regulations between NF-κB and HIF-1 have not been adequately addressed in previous research. Here, we report that hypoxia increased NF-κB in hepatocellular carcinoma cells. The HIF-1 protein level was rapidly induced by protein stabilization (by 2 hours) and then moderately decreased, whereas mRNA levels were reciprocally increased. We also found that NF-κB p50 and p65 (RelA), but not c-Rel, bound the HIF- 1a promoter, thus increasing its transcription. In contrast, miR-199a-5p and miR-93, c-Rel downstream targets, decreased HIF-1α at both the mRNA and protein levels. Dicer1, a key enzyme in miRNA biogenesis, was decreased by acute hypoxia but was later increased by HIF-1, rather than by the above-mentioned NF-κB subunits. Thus, NF-κB both positively and negatively fine-tuned HIF-1 in hypoxic hepatocarcinoma cells.
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Background: It is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.
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To further confirm the role of ST6Gal-I in the apoptosis of hepatocarcinoma cells, we used pcDNA3.1/ ST6Gal-I overexpression vector to upregulate the level of ST6Gal-I expression in Huh7 cells. A significant increase in ST6Gal-I expression at mRNA, protein and glycans levels after pcDNA3.1/ST6Gal-I transfection were observed by RT-PCR, Western-blot and Lectin-blot assays (*P<0.05) (Figure 4Ai, Aii and Aiii). By using Annexin V-FITC/PI and DAPI staining, we found that the apoptosis rates of ST6Gal-I overexpressed cells decreased statistically compared to control cells in the presence or absence of docetaxel (Figure 4B and 4C).In addition, we also found that ST6Gal-I overexpressed cells were more resistant to docetaxel than control cells when exposed to different concentrations for 24h and 48h (Figure 4D). Together, these results indicate that upregulation of ST6Gal-I could protect Huh7 cells from docetaxel-
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role in the cancer progress and has the potential for providing a novel thread for cancer therapy. In current study, we demonstrate the roles of GSN on anti-apoptosis of hepatocarcinoma cells by transcriptome RNA-seq method. Then flow cytometry (FCM), in-cell immunoblotting and transmission electron microscopy (TEM) were used to ex- amine the GSN regulatory cell apoptosis. The results revealed GSN significantly suppresses apoptosis-associated functional categories through down-regulating apoptosis-associated genes in 5 apoptosis terms and 6 relevant KEGG pathways. FCM showed a significant lower apoptotic rate in GSN-SMMC7721 (P<0.05). In-cell immunoblot- ting detected discrepant expression of the apoptosis factors among GSN expressed/shRNA transfectants (P<0.05). TEM observed the discernible apoptosis morphology. Above results suggest a negative relationship between GSN expression and hepatocarcinoma cell apoptosis. GSN overexpression suppresses apoptosis while down-regulated GSN promotes apoptosis. The possible mechanism could be associated with the regulation of GSN on the apoptosis- associated pathways and the apoptosis factors caspase 3 and bcl-2.
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MTT assays indicated that QNT11 inhibited the prolifer- ation of human hepatocarcinoma cells in a dose- and time-dependent manner but did not appear to disturb the proliferation of non-cancerous BMSCs. The result provided the evidence at QNT11 selectively suppresses cancer cell proliferation. Thus, we propose that QNT11 could be an effective candidate for therapy against ma- lignant tumors. Antibacterial fluoroquinolones are a class of antibacterial agents that are commonly used to treat human and animal infections. The treatment of bacterial infection inhibits bacterial DNA gyrase by a mechanism similar to that of certain antitumor drugs against mammalian topoisomerase II . Some antibac- terial fluoroquinolones, such as ciprofloxacin, ofloxacin and norfloxacin, also demonstrate a slight interaction with mammalian topoisomerase II, although these anti- bacterials are much more selective for bacterial DNA gyrase . However, a number of chemically modifed antibacterial fuoroquinolone derivatives with enhanced activity against mammalian topoisomerase II have been developed. Strong inhibitory effects against eukaryotic DNA replication were demonstrated, and their structure- activity relationship has also been characterized . These fluoroquinolone derivatives share a similar mechanism of
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Cell apoptosis analysis: Huh7, Huh7-HBx and Huh7-3 .1cells were plated and grown overnight until they reach 80, confluence, then the cells were treated with 1 μg/ml ADM or/and IMD-0354. Subsequently, detached cells in the medium were collected, and the remaining adherent cells were released by trypsinization. The cells were washed with phosphate-buffered saline (PBS) and resuspended in 250 μL binding buffer (annexinV-FITC kit; BECKMAN COULTER) containing 5 μL of annexin V-FITC stock and 10 μL of 20 μg/mL propidium iodide (PI). After incubated for 10 min at room temperature in a light protected area, the samples were analyzed by FACS (BECKMAN COULTER FC500 MPL) using muticycle software. We could discriminate intact cells (annexin - /PI - ) from apoptotic cells (annexin + /PI - & annexin + PI + ) and necrotic cells (annexin - /PI + ) after treatment with ADM.
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Sorafenib, a multikinase inhibitor, increases survival of patients with advanced hepatocellular carcinoma . In one study, median overall survival was 10.7 months in the sorafenib group and 7.9 months in the placebo group . For this reason, one of our objectives was to compare the effectiveness in vitro and in vivo of pravastatin for the treatment of hepatocarcinoma. We observed that the com- bination of pravastatin and sorafenib in vitro, considerably decreased cell proliferation and the expression of MAT1A in vivo. The results were confirmed in vivo. In particular, the combination of pravastatin and sorafenib resulted in a smaller number and size of hepatocarcinoma lesions, com- pared to the administration of the two drugs separately. As well as decreasing levels of PCNA and MAT1A, sorafenib also decreased the expression of Mcl-1 messenger RNA and protein, transcriptional targets of STAT3, as well as sensitizing neoplastic cells to tumour necrosis factor- related apoptosis-inducing ligand (TRAIL)-mediated apop- tosis . In addition, sorafenib produces inhibition of the expression of phospho-MEK, phospho-ERK, cyclin D1, Rb and anti-apoptotic proteins Bcl-xl and Mcl-1 [27,28]. U/L
Results obtained from this study indicate that 6 out of a total of 28 plants and 5 recipes (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, and Pra-Sa-Prao-Yhai recipe) used in Thai folklore medicine exhibited promising cytotoxic activity against CL-6 human cholangiocarcinoma cell line. Sensi- tivity to other cancerous cell lines varied according to cell types and the hepatocarcinoma HepG2 appears to be the most resistant cell line to the tested extracts. The extract from Atractylodes lancea appears to be both the most potent and most selective against cholangiocarci- noma, whereas that from Zingiber officinal appears to be the most potent and most selective against HepG2. The extract from Piper chaba (IC 50 = 18.63 μg/ml, SI =
SOD is specialized to convert highly superoxide radicals to less toxic H 2 O 2 . Higher production of intracellular ROS and membrane LPO in Co 3 O 4 NP-exposed cells along with depletion of antioxidant components suggest that oxida- tive stress might be the primary mechanism for toxicity of Co 3 O 4 NPs in HepG2 cells. In the present study, ROS and MDA levels were significantly higher, while the antioxidant GSH level was significantly lower in HepG2 cells exposed to Co 3 O 4 NPs. ROS, such as O 2- , OH• and H
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Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Advanced or recurrent HCC is frequently resistant to conventional chemotherapeutic agents and radiation. Therefore, targeted agents with tolerable toxicity are mandatory to improve HCC therapy and prognosis. In this neoplasia, the PI3K/Akt signaling network has been frequently shown to be aberrantly up-regulated. To evaluate whether Akt could represent a target for treatment of HCC, we studied the effects of the allosteric Akt inhibitor, MK-2206, on a panel of HCC cell lines characterized by different levels of Akt-1 activation. The inhibitor decreased cell viability and induced cell cycle arrest in the G 0 /G 1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1. Moreover, MK-2206 induced apoptosis, as documented by Annexin V labeling, and also caused autophagy, as evidenced by increased levels of the autophagy marker LC3A/B. Autophagy was shown to be a protective mechanism against MK-2206 cytotoxicity. MK-2206 down- regulated, in a concentration-dependent manner, the phosphorylation levels of Akt- 1 and its downstream targets, GSK3 α/β and FOXO3A. MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment. Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.
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Tumor cells (rat glioma, human lung giant carcinoma and human rhabdomyosarcoma) transfected with connexin 43 cDNA recovered GJIC capacity and they showed cell growth inhibition [11-13]. In the liver, these approaches have been less explored. At the moment, it is not clear whether the exogenous expression of Cxs would restore the gap junction and/or contribute to cell growth inhibi- tion. Eghbali et al. did not observe growth inhibition in Cx32 transfected cells, however when these cells were injected in animals they induced smaller tumors than the non-transfected cells. This discrepancy can be related to Cxs expression differential pattern observed "in vitro" and "in vivo" (Cx43 and Cx32, respectively). Considering that studies "in vitro" still represent an important tool in the liver cancer investigations and that Cx43 represents the major form expressed in normal liver cell lines and hepatoma cells, it would be interesting to further explore the relationship among the exogenous expression of Cx43, GJIC and proliferative behavior. The present work evaluated the effects of exogenous Cx43 expression on the
Heat shock proteins (HSP) form a family with a fundamental role in the correct folding and functionalization of proteins synthesized inside the cell. Clinical studies in patients with HCC show that the expression of HSP-60 is diminished in tumor cells with respect to healthy livers. This finding is related to a greater invasive ability of this neoplasm and a lower survival rate in patients. The lower differentiation gives these cells greater mobility, resulting in a high rate of invasion and subsequent metastasis . Our results show that uvaol treatment induces an increase in the expression of HSP- 60 in WRL68 control line, being this rise more acute for the HepG2 line. This effect of uvaol in HCC lines is a promising result, since it is related to the results obtained by Zhang et al. . These authors observed that after inducing an overexpression of the gene that codes for the HSP-60 in HepG2 cells, these cells developed a phenotype with lower migratory capacity and greater cell differentiation, thus decreasing their metastatic ability . Together with HSP-60 overexpression, wound healing results obtained in this study confirm the effect of uvaol as a potential anti-migratory compound.
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Finally, as in normoxia, we also found that autophagy protects HCC cells from the cytotoxic effects of BGT226. This was assessed by MTT assays, after treating SNU475 and Mahlavu cells with BGT226 0.5 μM and CQ 10 and 25 μM for 24 h. CQ alone displayed only limited cytotoxic effects against SNU475 and Mahlavu cells. However, when it was combined with BGT226, it was possible to detect an increased cytotoxicity in both cell lines (Figure 5C). To extend the analysis concerning the protective role of autophagy also in hypoxia we used the autophagy inhibitor 3-MA. The inhibitor alone did not affect cell growth of Mahlavu and Hep3B after 24 h of treatment. However, when it was combined with 0.5 μM BGT226, it was possible to detect an increased cytotoxicity in both cell lines (Figure 5D).
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Additional file 1: Figure S1. Solvent controls do not influence cell viability as well as intracellular NAMPT and NAD levels. HepG2 cells were stimulated with serum-free medium with and without DMSO (dilution factor: 1:1 ′ 000 ‘ 000) and serum free medium with 1% free fatty acid -BSA with and without NaOH (dilution 1:200). Neither DMSO nor NaOH altered cell viability measured by WST-1 assay (A), intracellular NAD levels measured by HPLC (B), NAMPT mRNA expression or protein abundance measured by qPCR or Western blot analysis (C,D), respectively, enzyme activity (E) and lipid accumulation stained with Oil-red O (F). Data were normalised to control (serum free medium) which was set 1. Data represent two or three independent experiments shown as means ± SEM. FFA: free fatty acid. (JPEG 691 kb)
Considering the important role of inflammatory cyto- kines in tumor development, we next analyzed whether exo-adipocytes affected HepG2 cells and provided a benefit for tumor growth by using a nude mouse xeno- graft tumor model. HepG2 cells were subcutaneously co-implanted with exo-adipocytes, adipocytes, or PBS at a ratio of 10:1. Coinjection with exo-adipocytes re- sulted in an increased tumor weight compared with tumor cells injected with adipocytes or PBS (Fig. 3a).To determine the effect of exo-adipocytes on angiogenesis and proliferation of tumor cells in vivo, we performed IHC staining to detect CD31 and Ki67. Coinjected exo-adipocytes could enhance the vascular density as demonstrated by the increased expression of CD31 (Fig. 3b,c). Figure 3d, e revealed that the numbers of Ki67-positive cells were increased significantly in the presence of exo-adipocytes. When the tumors were ex- cised for assessment of immune cell infiltration, we ob- served that the number of F4/80 macrophages was higher in tumors receiving exo-adipocytes than those receiving control adipocytes (Fig. 3f, g). Previous studies suggest IL-6 to be a major regulator of tumor-stroma interaction in cancer microenvironment . Here, we examined IL-6 expression levels in tumor sections and found increased IL-6 protein levels (Fig. 3h, i), consistent with the upregulation of IL-6 genes shown in Fig. 2e. In addition, we observed the appearance of adipocytes among cancer cells in the tumor sections, suggesting that adipocytes were not consumed by neighboring cancer cells during the 4-week tumorigenesis process (Fig. 3j). Taken together, exo-adipocytes were endowed with a capability by tumor exosomes to promote tumor growth, enhance angiogen- esis, and recruit macrophages in vivo.
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perature. Endogenous peroxidase activity was blocked with peroxidase-blocking reagent con- taining 3% hydrogen peroxide and serum. After incubation, the slides were washed in PBS 3 times. Then, slides were incubated with P62 polyclonal antibody (1:100; Proteintech Inc., USA) overnight at 4°C. After being washed, the sections were incubated with biotin-goat anti- rabbit antibody at room temperature. The per- oxidase reaction was developed with 3,3’-diami- nobenzidine (DAB) chromogen solution in DAB buffer substrate. The sections were counter- stained with hematoxylin, mounted in neutral gum, and analyzed using a bright field micro- scope (Olympus IX71, Tokyo, Japan). The immu- nostaining was microscopically evaluated by two independent pathologists. A semiquantita- tive scoring system was based on the staining intensity and proportion of positive cells. Western blotting assay