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Sample records for hepatocarcinoma humano hep3b

  1. Caffeic acid phenethyl ester enhances TRAIL-mediated apoptosis via CHOP-induced death receptor 5 upregulation in hepatocarcinoma Hep3B cells.

    PubMed

    Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Park, Sang Rul; Choi, Yung-Hyun; Choi, Il-Whan; Kim, Gi-Young

    2016-07-01

    Caffeic acid phenethyl ester (CAPE) exhibits various pharmaceutical properties, including anti-bacterial, anti-inflammatory, anti-viral, anti-cancer, and anti-oxidative activity. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been a promising anti-cancer agent that preferentially induces cancer cell apoptosis with negligible cytotoxicity toward normal cells. Therefore, the present study investigated whether CAPE promotes TRAIL-mediated cytotoxicity in hepatocarcinoma Hep3B cells. The present study demonstrated that CAPE sensitized TRAIL-mediated cell death in Hep3B carcinoma cells. The percentages of the apoptotic cells and annexin-V(+) cells significantly increased in combined treatment with CAPE and TRAIL (CAPE/TRAIL). Treatment with pancaspase inhibitor, z-VAD-fmk, attenuated CAPE/TRAIL-induced apoptosis, suggesting that the combined treatment triggers caspase-dependent apoptosis. Additionally, we found that CAPE stimulated the expression of death receptor 5 (DR5) and treatment with DR5/Fc chimera protein significantly blocked CAPE/TRAIL-induced apoptosis, which indicates that CAPE/TRAIL stimulated apoptosis through the binding of TRAIL to DR5. Moreover, expression of transcription factor C/EBP homologous protein (CHOP) markedly increased in response to CAPE and transient knockdown of CHOP abolished CAPE/TRAIL-mediated apoptosis. These results suggest that CHOP is a key regulator in CAPE/TRAIL-mediated apoptosis. Taken together, the present study found that CAPE significantly enhanced TRAIL-mediated apoptosis in Hep3B carcinoma cells and suggested that CAPE has promising potential in chemoprevention of hepatocellular carcinomas. PMID:27260301

  2. Dieckol, isolated from Ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma Hep3B cells.

    PubMed

    Yoon, Jin-Soo; Kasin Yadunandam, Anandam; Kim, Soon-Jin; Woo, Hee-Chul; Kim, Hyeung-Rak; Kim, Gun-Do

    2013-07-01

    Phlorotannins have been reported to demonstrate several biological properties, including antioxidant activity, and activities useful in the treatment of diabetic complications and in chemoprevention of several vascular diseases. In this study, we focused on the apoptosis induced by dieckol, a marine algal phlorotannin isolated from Ecklonia stolonifera, on human hepatocellular carcinoma (HCC) Hep3B cells. Dieckol reduced the numbers of viable cells and increased the numbers of apoptotic cells in a dose-dependent manner. Immunoblotting analysis revealed that dieckol increased the expression levels of cleaved caspases-3, 7, 8, and 9, and cleaved poly(ADP-ribose) polymerase. Dieckol increased the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with apoptosis-inducing factor. In addition, dieckol induced increased expression of truncated Bid and Bim. The results indicate that dieckol induces apoptosis via the activation of both death receptor and mitochondrial-dependent pathways in HCC Hep3B cells. PMID:23054486

  3. Differential effects of phorbol ester on growth and protein kinase C isoenzyme regulation in human hepatoma Hep3B cells.

    PubMed Central

    Hsu, S L; Chou, Y H; Yin, S C; Liu, J Y

    1998-01-01

    PMA has both mitogenic and antiproliferative effects on human hepatoma Hep3B cells. In response to low PMA concentration (10 nM), Hep3B cells displayed an increasing proliferation potentiation. At high PMA concentration (1 microM) Hep3B cells exhibited modest cytostatic effects. Determinations of protein kinase C (PKC) activity in PMA-treated cells revealed that alterations in PKC activity are associated with proliferative capacity. The decrease in PKC activity mediated by a high dose of PMA was accompanied by cell growth inhibition. Increases in PKC activity mediated by a low dose of PMA were consistent with proliferation stimulation. Immunoblot analysis showed that there are at least six PKC isoenzymes: alpha, delta, epsilon, mu, zeta and iota/lambda, constitutively expressed in Hep3B cells. Cellular fractionation and immunocytochemical staining results demonstrated that both 10 nM and 1 microM PMA treatments induced a marked translocation of PKC-alpha from cytosol to membrane or nuclear fraction within 5-30 min. At the same time PKC-delta and epsilon were translocated from the membrane to nuclear fraction. In addition, prolonged treatment with 1 microM PMA, but not with 10 nM PMA, selectively mediated the down-regulation of these three PKC isoenzymes. The distinct effects of different concentrations of PMA on cell proliferation and PKC-alpha, delta and epsilon isoenzyme modulation support the involvement of these three PKC isotypes in the mechanism of action of Hep3B cells in cell growth events. PMID:9639562

  4. The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

    SciTech Connect

    Yan, Chunlan; Oh, Joon Seok; Yoo, Seung Hee; Lee, Jee Suk; Yoon, Young Geol; Oh, Yoo Jin; Jang, Min Seok; Lee, Sang Yeob; Yang, Jun; Lee, Sang Hwa; Kim, Hye Young; Yoo, Young Hyun

    2013-01-01

    Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1-independent

  5. Cell Cycle Regulation in the Estrogen Receptor Beta (ESR2)-Overexpressing Hep3B Hepatocellular Carcinoma Cell Line.

    PubMed

    Liu, Yi-Sheng; Tsai, Ying-Lan; Yeh, Yu-Lan; Chung, Li-Chin; Wen, Su-Ying; Kuo, Chia-Hua; Lin, Yueh-Min; Padma, V Vijaya; Kumar, V Bharath; Huang, Chih-Yang

    2015-04-30

    Epidemiological studies and experimental data have shown that the incidences of hepatocellular carcinoma in men are more frequent than in women. Evidence suggests that imbalance of hormones, including estrogen, androgen, prolactin, and growth hormone, modifies liver tumorigenesis. In this present study, we investigated how estrogen and estrogen receptor 2 (ESR2), regulates the cell cycle mechanism in Hep3B hepatocellular carcinoma cell line. Our results showed that ESR2 overexpression in the presence of 10⁻⁸ M 17-β-estradiol downregulated c-myc and cyclin D1 expression and simultaneously upregulated p27 expression. However, flow cytometry and MTT assays showed only minor G₁ phase arrest without affecting cell viability. Taken together, these observations indicate that ESR2 is required to lower tumorigenesis in males by altering cell cycle proteins in a ligand-dependent manner. PMID:25858474

  6. Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms.

    PubMed

    Charette, N; De Saeger, C; Horsmans, Y; Leclercq, I; Stärkel, P

    2013-01-01

    Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma. PMID:23348585

  7. Antiproliferative activity of Humulus lupulus extracts on human hepatoma (Hep3B), colon (HT-29) cancer cells and proteases, tyrosinase, β-lactamase enzyme inhibition studies.

    PubMed

    Cömert Önder, Ferah; Ay, Mehmet; Aydoğan Türkoğlu, Sümeyye; Tura Köçkar, Feray; Çelik, Ayhan

    2016-01-01

    The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72 h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6-1 mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1 mg/mL for 72 h in Hep3B cells and 0.1-0.2 mg/mL for 48 and 72 h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (V(max)) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and β-lactamase (penicillinase). PMID:25683080

  8. Inactivated Tianjin strain, a novel genotype of Sendai virus, induces apoptosis in HeLa, NCI-H446 and Hep3B cells

    PubMed Central

    CHEN, JUN; HAN, HAN; WANG, BIN; SHI, LIYING

    2016-01-01

    The Sendai virus strain Tianjin is a novel genotype of the Sendai virus. In previous studies, ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) demonstrated antitumor effects on human breast cancer cells. The aim of the present study was to investigate the in vitro antitumor effects of UV-Tianjin on the human cervical carcinoma HeLa, human small cell lung cancer NCI-H446 and human hepatocellular carcinoma Hep 3B cell lines, and the possible underlying mechanisms of these antitumor effects. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that UV-Tianjin treatment inhibited the proliferation of HeLa, NCI-H446 and Hep 3B cells in a dose- and time-dependent manner. Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide double staining indicated that UV-Tianjin induced dose-dependent apoptosis in all three cell lines with the most significant effect observed in the HeLa cell line. In the HeLa cell line, UV-Tianjin-induced apoptosis was further confirmed by the disruption of the mitochondria membrane potential and the activation of caspases, as demonstrated by fluorescent cationic dye and colorimetric assays, respectively. In addition, western blot analysis revealed that UV-Tianjin treatment resulted in significant upregulation of cytochrome c, apoptosis protease activating factor-1, Fas, Fas ligand and Fas-associated protein with death domain, and activated caspase-9, −8 and −3 in HeLa cells. Based on these results, it is hypothesized that UV-Tianjin exhibits anticancer activity in HeLa, NCI-H446 and Hep 3B cell lines via the induction of apoptosis. In conclusion, the results of the present study indicate that in the HeLa cell line, intrinsic and extrinsic apoptotic pathways may be involved in UV-Tianjin-induced apoptosis. PMID:27347098

  9. The protective effects of epigallocatechin gallate on lipopolysa ccharide-induced hepatotoxicity: an in vitro study on Hep3B cells

    PubMed Central

    Karamese, Murat; Guvendi, Bulent; Karamese, Selina Aksak; Cinar, Irfan; Can, Serpil; Erol, Huseyin Serkan; Aydin, Hakan; Gelen, Volkan; Karakus, Emre

    2016-01-01

    Objective(s): In the present study, our aim was to investigate the possible protective effects of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS)-induced hepatotoxicity by using Hep3B human hepatoma cells. Specifically, the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of LPS-associated cytotoxicity. Consequently, the hepatocellular carcinoma cell line Hep3B was chosen as a model for investigation of LPS toxicity and the effect of EGCG on LPS-exposed cells. Materials and Methods: The Hep3B human hepatoma cells were used for this study. The cytotoxic effects of chemicals (EGCG and LPS), AST and ALT levels, SOD and CAT activities, GSH-Px level and TNF-alpha and IL-6 levels were detected by using different biochemical and molecular methods. LPS and EGCG were applied to cells at various times and doses. Results: The highest treatment dose of EGCG (400 µM) led to a dramatic decrease in SOD level and increase in CAT and GSH levels. Additionally, the highest dose of EGCG also led to a dramatic increase in TNF-alpha and IL-6 levels. On the other hand, effective doses of EGCG (200 and 100 µM) normalized all related parameters levels. Conclusion: LPS caused hepatotoxicity, but interestingly, a high dose of EGCG was found to be a cytotoxic agent in this study. However, other two doses of EGCG led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels. Further studies should examine the effect of EGCG on secondary cellular signaling pathways. PMID:27403254

  10. A novel cantharidin analog N-Benzylcantharidinamide reduces the expression of MMP-9 and invasive potentials of Hep3B via inhibiting cytosolic translocation of HuR

    SciTech Connect

    Lee, Ji-Yeon; Chung, Tae-Wook; Choi, Hee-Jung; Lee, Chang Hyun; Eun, Jae Soon; Han, Young Taek; Choi, Jun-Yong; Kim, So-Yeon; Han, Chang-Woo; Jeong, Han-Sol; Ha, Ki-Tae

    2014-05-02

    Highlights: • We examined the inhibition of N-Benzylcantharidinamide on MMP-9-mediated invasion. • Unlike cantharidin, N-Benzylcantharidinamide has very low toxicity on Hep3B cells. • The reduced MMP-9 expression was due to HuR-mediated decrease of mRNA stability. • We suggest N-Benzylcantharidinamide as a novel inhibitor of MMP-9-related invasion. - Abstract: Invasion and metastasis are major causes of malignant tumor-associated mortality. The present study aimed to investigate the molecular events underlying inhibitory effect of N-Benzylcantharidinamide, a novel synthetic analog of cantharidin, on matrix metalloproteinase-9 (MMP-9)-mediated invasion in highly metastatic hepatocellular carcinoma Hep3B cells. In this investigation, among six analogs of cantharidin, only N-Benzylcantharidinamide has the inhibitory action on MMP-9 expression at non-toxic dose. The MMP-9 expression and invasion of Hep3B cells were significantly suppressed by treatment of N-Benzylcantharidinamide in a dose-dependent manner. On the other hand, the transcriptional activity of MMP-9 promoter and nuclear levels of NF-κB and AP-1 as the main transcriptional factors inducing MMP-9 expression were not affected by it although the level of MMP-9 mRNA was reduced by treatment of N-Benzylcantharidinamide. Interestingly, the stability of MMP-9 mRNA was significantly reduced by N-Benzylcantharidinamide-treatment. In addition, the cytosolic translocation of human antigen R (HuR), which results in the increase of MMP-9 mRNA stability through interaction of HuR with 3′-untranslated region of MMP-9 mRNA, was suppressed by treatment of N-Benzylcantharidinamide, in a dose-dependent manner. Taken together, it was demonstrated, for the first time, that N-Benzylcantharidinamide suppresses MMP-9 expression by reducing HuR-mediated MMP-9 mRNA stability for the inhibition of invasive potential in highly metastatic Hep3B cells.

  11. Evaluation of the therapeutic efficacy of tripeptide tyroserleutide (YSL) for human hepatocarcinoma by in vivo hollow fiber assay.

    PubMed

    Li, Xiao-lei; Liu, Jun-yan; Lu, Rong; Xu, Qiong; Zhu, Zhi-feng; Wang, Li; Zhou, Chun-lei; Jia, Jing; Fu, Zheng; Yao, Zhi

    2008-12-01

    Tyroserleutide (YSL), extracted from spleen of pigs, is a tripeptide that has shown therapeutic efficacy in an experimental BEL-7402 human hepatocarcinoma model. The hollow fiber assay (HFA) is a solid tumor model for large-scale screening of potential anticancer compounds that minimizes expenditures of materials, time, and money. Tumor cells are cultivated within biocompatible, semipermeable hollow fibers, which are implanted in immunosuppressed mice. In this study, the HFA was used to investigate the therapeutic efficacy of YSL for human hepatocarcinoma. In vitro effects of YSL on human hepatocarcinoma cell lines BEL-7402, SMMC-7721, Hep3B, HepG2, and SK-HEP-1 were assayed by the MTS method. In vivo effects of YSL on the five human hepatocarcinoma cell lines were assayed by HFA. Mice implanted with tumor cells in hollow fibers were treated with YSL, and the effects of YSL on tumor cell populations were assessed by MTT assay. YSL significantly inhibited the proliferation of the five human hepatocarcinoma, both in vitro and in vivo (P < 0.05). The HFA is a rapid, accurate, and economical method for evaluating the inhibitory effects of drugs on different tumor cells in vivo. These results support the clinical application of YSL for treatment of human hepatocarcinoma. PMID:18264678

  12. Anti-proliferative and apoptosis-triggering potential of disulfiram and disulfiram-loaded polysorbate 80-stabilized PLGA nanoparticles on hepatocellular carcinoma Hep3B cell line.

    PubMed

    Hoda, Muddasarul; Pajaniradje, Sankar; Shakya, Garima; Mohankumar, Kumaravel; Rajagopalan, Rukkumani

    2016-08-01

    There is an emerging trend to restudy known drugs for their anti-cancer potential. One such anti-alcoholic drug, disulfiram, with significant anti-cancer potential was studied for its efficacy against Hep3B cell lines, an in vitro model of hepatocellular carcinoma. Simultaneously, we intended to study the effect of polysorbate 80-stabilized PLGA nanoparticles and its DSF-loaded counterpart. Cell and nuclear staining, comet assay, flow cytometry and Western blots were performed. Results suggest that cell proliferation was inhibited by DSF and its PLGA nanoparticles through cell cycle arrest, triggering activation of apoptotic pathways that culminates with cell death. DSF loaded nanoparticles when compared with free DSF, showed significantly lesser effect due to its sustained drug-releasing property, while empty nanoparticles showed negligible influence on Hep3B cells. Our results suggest that DSF alone contributes to cell death, while polysorbate 80-stabilized PLGA nanoparticles show sustained drug release patterns that would potentially lower dosage regimens. PMID:27013133

  13. 4,4'-Diaminodiphenyl ether derivatives: synthesis, spectral, optical, thermal characterization and in-vitro cytotoxicity against Hep 3B and IMR 32 human cell lines.

    PubMed

    Singh, Vinay K; Kadu, Rahul; Roy, Hetal

    2014-03-01

    4,4'-Diaminodiphenyl ether was selected as a lead compound to prepare a novel series of bisimine derivatives bearing polyaromatic hydrocarbon substituents and their reduced benzyl forms. The new compounds were structurally characterized by microanalysis, mass, IR, (1)H, (13)C, DEPT-135, HSQC, g-COSY NMR spectroscopy, UV-visible, fluorescence spectrophotometers and by thermogravimetric analysis. The antitumor activity of these derivatives was evaluated in-vitro against Hep 3B and IMR 32 by the MTT assay and the results were compared with cisplatin. Interestingly, some compounds were found extremely active against both the cell lines and proved to be more potent as cytotoxic agents than cisplatin. Morphological evidences suggest the induction of apoptosis and explain the mode of action of these derivatives as antitumor agents. PMID:24531196

  14. Characterization of UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B.

    PubMed

    Park, C; Jin, U H; Lee, Y C; Cho, T J; Kim, C H

    1999-07-15

    UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1, 6-N-acetylglucosaminyltransferase V (GlcNAcT-V) has been purified from cell extracts of the human hepatoma cell line, Hep3B, with 8.7% recovery. The purified enzymes had molecular masses of about 67 and 65 kDa on denaturated and natural conditions, respectively. The values of pI was 5.9. The GlcNAcT-V, when resolved by SDS-PAGE, was positive for Schiff staining, suggesting that the enzyme is glycoprotein. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzyme displayed a temperature optimum of around 50 degrees C and optimum an pH of 6.5. The enzyme was stable in response to incubation from pH 4.5 to pH 10.5 at 4 degrees C for 24 h. The presence of UDP-GlcNAc and GlcN,GlcN-bi-PA protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ion; however, it was inhibited by Fe3+. The enzyme activity was inhibited by another series of NDP-sugars including ADP-, CDP-, GDP-, and TDP-GlcNAc. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward biantennary (GlcN,GlcN-bi-PA) sugars. The enzymes had apparent Km values of 1.28 and 5.8 mM for GlcN,GlcN-bi-PA and UDP-GlcNAc, respectively. In order to isolate the GlcNAcT-V gene, PCR primers of GNN-1 and GNN-8 were designed and the amplified PCR product carrying the gene was cloned and sequenced. Nucleotide sequence analysis showed a 2220-bp open reading frame encoding a 740-amino-acid protein. This was almost same as the previously reported human sequences, except for some sequence differences in three amino acids. The three amino acid changes were as follows: 375V --> L, 555T --> R, and 592A --> G. These studies represent the detailed characterization of a purified GlcNAcT-V from human hepatoma cell Hep3B. PMID:10395745

  15. IL-1 receptor antagonist affects the plasma protein response of Hep 3B cells to conditioned medium from lipopolysaccharide-stimulated monocytes.

    PubMed

    Damtew, B; Rzewnicki, D; Lozanski, G; Kushner, I

    1993-05-01

    The availability of the IL-1R antagonist (IL-1ra) has made it possible to assess the specific contributions of IL-1 to the acute phase changes induced by complex mixtures of cytokines. We utilized IL-1ra to define the contribution of IL-1 to the effects of conditioned medium from LPS-stimulated monocytes on production of the positive acute phase proteins C-reactive protein, serum amyloid A, fibrinogen, alpha 1-protease inhibitor, complement component C3, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, and ceruloplasmin and the negative acute phase proteins albumin and transferrin in Hep 3B cells. Induction of C-reactive protein and serum amyloid A was essentially abolished, induction of complement component C3 and alpha 1-acid glycoprotein was moderately decreased and induction of fibrinogen was enhanced. In contrast, there was no significant effect of IL-1ra on induction by conditioned medium of alpha 1-protease inhibitor, alpha 1-antichymotrypsin, or ceruloplasmin. IL-1ra partially blocked the down-regulatory effects of conditioned medium on both of the negative acute phase proteins we studied--albumin and transferrin. These findings enhance our understanding of the contribution of IL-1 to the acute phase response. In addition, they indicate that IL-1ra in vivo may influence synthesis of both positive and negative acute phase proteins. PMID:7682588

  16. FK866-induced NAMPT inhibition activates AMPK and downregulates mTOR signaling in hepatocarcinoma cells

    SciTech Connect

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Gebhardt, Rolf; Weiss, Thomas S.; Kiess, Wieland; Garten, Antje

    2015-03-06

    Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. - Highlights: • FK866 increases cell death in p53-deficient hepatocarcinoma cells. • AMPK/mTOR signaling is dysregulated in hepatocarcinoma cells. • FK866-induced NAMPT inhibition activates AMPK

  17. Differential Antitumoral Properties and Renal-Associated Tissue Damage Induced by Tacrolimus and Mammalian Target of Rapamycin Inhibitors in Hepatocarcinoma: In Vitro and In Vivo Studies

    PubMed Central

    Pereira, Sheila; Wang, Jize; Aliseda, Sara; Rodríguez-Hernández, María A.; González, Raúl; Marín-Gómez, Luís M.; Gómez-Bravo, Miguel A.; Padillo, Francisco J.; Álamo-Martínez, José M.; Muntané, Jordi

    2016-01-01

    Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with potential portal hypertension and/or bilirubinemia, but without vascular-associated diseases. The patients are receiving immunosuppressive therapy to reduce graft rejection, but differential side effects have been related to calcineurin and mTOR inhibitor administration regarding tumor recurrence and nephrotoxicity. The in vitro studies showed that Tacrolimus exerted a more potent pro-apoptotic effect than Everolimus (Huh 7>Hep 3B>HepG2), being sirolimus only active in Hep3B cell line. Tacrolimus and Everolimus exerted potent antiproliferative properties in Huh 7 and Hep3B in which cells Sirolimus was inactive. Interestingly, Tacrolimus- and Everolimus-dependent G0/G1 cell accumulation occurred as a consequence of drastic reduction in S, as well as in S and G2+M phases, respectively. The in vivo studies support data on the more effective antitumoral properties of Everolimus, eventual risk of pro-angiogenic tumoral properties and nephrotoxicity of Tacrolimus, and pro-proliferative properties of Sirolimus in tumors developed in nude mice. PMID:27518575

  18. Inhibition of doxorubicin-induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib--the role of extracellular signal-regulated kinase counteraction.

    PubMed

    Manov, Irena; Pollak, Yulia; Broneshter, Rinata; Iancu, Theodore C

    2011-09-01

    A multikinase inhibitor of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects. PMID:21790999

  19. Discovery of a novel anti-cancer agent targeting both topoisomerase I and II in hepatocellular carcinoma Hep 3B cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

    PubMed

    Perng, Daw-Shyong; Tsai, Yu-Hsin; Cherng, Jonathan; Kuo, Chih-Wei; Shiao, Chih-Chung; Cherng, Jaw-Ming

    2016-08-01

    Cinnamomum verum has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, in hepatocellular carcinoma Hep 3B cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an up-regulation of pro-apoptotic bax and bak genes and down-regulation of anti-apoptotic bcl-2 and bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase 3 and 9, increase in the DNA content in sub G1, and morphological characteristics of apoptosis. 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments (VAC), suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against Hep 3B cells is accompanied by downregulations of NF-κB binding activity, inflammatory responses involving COX-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26707867

  20. Effects of shRNA-Mediated SOX9 Inhibition on Cell Proliferation and Apoptosis in Human HCC Cell Line Hep3B Mediated by Ultrasound-Targeted Microbubble Destruction (UTMD).

    PubMed

    Xue, Yan; Yang, Guilun; Wang, Changmei; Li, Xiaodong; Du, Guijin

    2015-11-01

    Hepatocellular carcinoma (HCC) is one of the most aggressive tumors in humans. The survival rate of patients is still very poor as current therapies offer limited treatment efficacy. Therefore, it is necessary to explore novel and more effective strategies to treat HCC. Recently, Ultrasound-targeted microbubble destruction (UTMD) has been shown to be a better alternative to viral vectors in delivering plasmid DNA into cells. In this study, we thus first determined the effect of combining UTMD with effectene on the transfection efficiency in human Hep3B cells. Transfection rate of the [effectene + shRNA-SOX9 + UTMD] group was the highest among the five groups, and were significantly higher than that of the [effectene + shRNA-SOX9] or [shRNA-SOX9 + UTMD] groups, while there was no significant difference between [shRNA-SOX9 alone] and [shRNA-SOX9 + UTMD] groups. Expression of SOX9 mRNA and protein was the lowest in effectene + shRNA-SOX9 + UTMD group. Moreover, transfection of shRNA-SOX9 with UTMD and effectene in combination could markedly inhibit the proliferation and induced cell apoptosis of Hep3B cells. These results suggest that the efficiency of gene delivery is remarkably increased when UTMD is combined with other transfection strategies, such as effectene. In conclusion, our research demonstrates that combining conventional transfection methods with UTMD achieves better transfection efficiency and that this can provide an improved gene delivery system for gene therapy. PMID:27352352

  1. Raf/ERK/Nrf2 signaling pathway and MMP-7 expression involvement in the trigonelline-mediated inhibition of hepatocarcinoma cell migration

    PubMed Central

    Liao, Jung Chun; Lee, Kun Tsung; You, Bang Jau; Lee, Chia Lin; Chang, Wen Te; Wu, Yang Chang; Lee, Hong-Zin

    2015-01-01

    Background Trigonelline occurs in many dietary food plants and has been found to have anti-carcinogenic activity. Trigonelline is also found in coffee which is one of the most widely consumed beverages. Many epidemiological studies have reported that coffee consumption has an inverse relationship with the risk of cirrhosis or hepatocellular carcinoma. It would be interesting to investigate whether trigonelline is an ideal chemoprevent agent to prevent cancer progression. Methods The protein expression was performed by western blotting. The trigonelline content in snow pea (Pisum sativum) was analyzed by high-performance liquid chromatography (HPLC). The migratory activity of human hepatocarcinoma cells (Hep3B) was assessed by using a wound migration assay. The percentage of each phase in the cell cycle was analyzed on a FACScan flow cytometer. Gene expression was detected by real-time reverse transcriptase-polymerase chain reaction techniques. Native gel analysis was performed to analyze the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. Results According to the data of HPLC analysis, P. sativum, which is a popular vegetable, has relatively high content of trigonelline. Our findings suggest that trigonelline is an efficient compound for inhibiting Hep3B cell migration. Trigonelline inhibited the migration of hepatoma cells at concentrations of 75–100 µM without affecting proliferation. Raf/ERK/Nrf2 protein levels and further downstream antioxidative enzymes activity, such as SOD, catalase, and glutathione peroxidase, significantly decreased after treatment with 100 µM of trigonelline for 24 h. The migration inhibition of trigonelline is also related to its ability to regulate the matrix metalloproteinases 7 (MMP-7) gene expression. Conclusions In this study, protein kinase Cα (PKCα) and Raf/ERK/Nrf2 signaling pathway and MMP-7 gene expression were involved in the trigonelline-mediated migration inhibition of Hep3B cells. We also

  2. Cytotoxic, apoptotic, and sensitization properties of ent-kaurane-type diterpenoids from Croton tonkinensis Gagnep on human liver cancer HepG2 and Hep3b cell lines.

    PubMed

    Pham, Minh Quan; Iscache, Anne Laure; Pham, Quoc Long; Gairin, Jean Edouard

    2016-04-01

    Human hepatocellular carcinoma (HCC) is the most common type of liver cancer, the second most common cause of death from cancer worldwide. A very poor prognosis and a lack of effective treatments make liver cancer a major public health problem, notably in less developed regions, particularly in eastern Asia. This fully justifies the search of new molecules and therapeutic strategies against HCC. Ent-kaurane diterpenoids are natural compounds displaying a broad spectrum of potential therapeutic effects including anticancer activity. In this study, we analyzed the pharmacological properties of a family of ent-kaurane diterpenoids from Croton tonkinensis Gagnep in human HepG2 and Hep3b cell lines, used as cellular reference models for in vitro evaluation of new molecules active on HCC. A structure-related cytotoxicity was observed against both HCC cell lines, enlighting the role of the 16-en-15-one skeleton of ent-kaurane diterpenoids. Cytotoxicity was closely correlated to apoptosis, evidenced by concentration-dependent subG1 cell accumulation, and increased annexin V expression. In addition, subtoxic concentration of ent-kaurane diterpenoid dramatically enhanced the sensitivity of HCC cells to doxorubicin. All together, our data bring strong support to the potential interest of ent-kaurane diterpenoids, alone or in combination with a cytotoxic agent, in cancer and more precisely against HCC. PMID:26713517

  3. Traditional Chinese medicine for prevention and treatment of hepatocarcinoma: From bench to bedside

    PubMed Central

    Hu, Bing; Wang, Shuang-Shuang; Du, Qin

    2015-01-01

    Traditional Chinese medicine (TCM) has played a positive role in the management of hepatocarcinoma. Hepatocarcinoma patients may present Qi-stagnation, damp-heat, blood stasis, Qi-deficiency, Yin-deficiency and other TCM syndromes (Zheng). Modern treatments such as surgery, transarterial chemoembolization (TACE) and high intensity focus ultrasound treatment would influence the manifestation of TCM syndromes. Herbs with traditional efficacy of tonifying Qi, blood and Yin, soothing liver-Qi stagnation, clearing heat and detoxifying and dissolving stasis, have been demonstrated to be potent to prevent hepatocarcinogenesis. TCM has been widely used in all aspects of integrative therapy in hepatocarcinoma, including surgical resection, liver transplantation, TACE, local ablative therapies and even as monotherapy for middle-advanced stage hepatocarcinoma. Clinical practices have confirmed that TCM is effective to alleviate clinical symptoms, improve quality of life and immune function, prevent recurrence and metastasis, delay tumor progression, and prolong survival time in hepatocarcinoma patients. The effective mechanism of TCM against hepatocarcinoma is related to inducing apoptosis, autophagy, anoikis and cell senescence, arresting cell cycle, regulating immune function, inhibiting metastasis and angiogenesis, reversing drug resistance and enhancing effects of chemotherapy. Along with the progress of research in this field, TCM will contribute more to the prevention and treatment of hepatocarcinoma. PMID:26019736

  4. Phenotypic Characterization Analysis of Human Hepatocarcinoma by Urine Metabolomics Approach

    PubMed Central

    Liang, Qun; Liu, Han; Wang, Cong; Li, Binbing

    2016-01-01

    Hepatocarcinoma (HCC) is one of the deadliest cancers in the world and represents a significant disease burden. Better biomarkers are needed for early detection of HCC. Metabolomics was applied to urine samples obtained from HCC patients to discover noninvasive and reliable biomarkers for rapid diagnosis of HCC. Metabolic profiling was performed by LC-Q-TOF-MS in conjunction with multivariate data analysis, machine learning approaches, ingenuity pathway analysis and receiver-operating characteristic curves were used to select the metabolites which were used for the noninvasive diagnosis of HCC. Fifteen differential metabolites contributing to the complete separation of HCC patients from matched healthy controls were identified involving several key metabolic pathways. More importantly, five marker metabolites were effective for the diagnosis of human HCC, achieved a sensitivity of 96.5% and specificity of 83% respectively, could significantly increase the diagnostic performance of the metabolic biomarkers. Overall, these results illustrate the power of the metabolomics technology which has the potential as a non-invasive strategies and promising screening tool to evaluate the potential of the metabolites in the early diagnosis of HCC patients at high risk and provides new insight into pathophysiologic mechanisms. PMID:26805550

  5. Iron depletion enhances the effect of sorafenib in hepatocarcinoma.

    PubMed

    Urano, Shinichi; Ohara, Toshiaki; Noma, Kazuhiro; Katsube, Ryoichi; Ninomiya, Takayuki; Tomono, Yasuko; Tazawa, Hiroshi; Kagawa, Shunsuke; Shirakawa, Yasuhiro; Kimura, Fumiaki; Nouso, Kazuhiro; Matsukawa, Akihiro; Yamamoto, Kazuhide; Fujiwara, Toshiyoshi

    2016-06-01

    ABSTACT Human hepatocellular carcinoma (HCC) is known to have a poor prognosis. Sorafenib, a molecular targeted drug, is most commonly used for HCC treatment. However, its effect on HCC is limited in clinical use and therefore new strategies regarding sorafenib treatment are required. Iron overload is known to be associated with progression of chronic hepatitis and increased risk of HCC. We previously reported that iron depletion inhibited cancer cell proliferation and conversely induced angiogenesis. Indeed iron depletion therapy including iron chelator needs to be combined with anti-angiogenic drug for its anti-cancer effect. Since sorafenib has an anti-angiogenic effect by its inhibitory targeting VEGFR, we hypothesized that sorafenib could complement the anti-cancer effect of iron depletion. We retrospectively analyzed the relationship between the efficacy of sorafenib and serum iron-related markers in clinical HCC patients. In clinical cases, overall survival was prolonged in total iron binding capacity (TIBC) high- and ferritin low-patients. This result suggested that the low iron-pooled patients, who could have a potential of more angiogenic properties in/around HCC tumors, could be adequate for sorafenib treatment. We determined the effect of sorafenib (Nexavar®) and/or deferasirox (EXJADE®) on cancer cell viability, and on cell signaling of human hepatocarcinoma HepG2 and HLE cells. Both iron depletion by deferasirox and sorafenib revealed insufficient cytotoxic effect by each monotherapy, however, on the basis of increased angiogenesis by iron depletion, the addition of deferasirox enhanced anti-proliferative effect of sorafenib. Deferasirox was confirmed to increase vascular endothelial growth factor (VEGF) secretion into cellular supernatants by ELISA analysis. In in vivo study sorafenib combined with deferasirox also enhanced sorafenib-induced apoptosis. These results suggested that sorafenib combined with deferasirox could be a novel combination

  6. Mobile phone radiation alters proliferation of hepatocarcinoma cells.

    PubMed

    Ozgur, Elcin; Guler, Goknur; Kismali, Gorkem; Seyhan, Nesrin

    2014-11-01

    This study investigated the effects of intermittent exposure (15 min on, 15 min off for 1, 2, 3, or 4 h, at a specific absorption rate of 2 W/kg) to enhanced data rates for global system for mobile communication evolution-modulated radiofrequency radiation (RFR) at 900- and 1,800-MHz frequencies on the viability of the Hepatocarcinoma cells (Hep G2). Hep G2 cell proliferation was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cell injury was evaluated by analyzing the levels of lactate dehydrogenase (LDH) and glucose released from lysed cells into the culture medium. Morphological observation of the nuclei was carried out by 4',6-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. In addition, TUNEL assay was performed to confirm apoptotic cell death. It was observed that cell viability, correlated with the LDH and glucose levels, changed according to the frequency and duration of RFR exposure. Four-hour exposure produced more pronounced effects than the other exposure durations. 1,800-MHz RFR had a larger impact on cell viability and Hep G2 injury than the RFR at 900 MHz. Morphological observations also supported the biochemical results indicating that most of the cells showed irregular nuclei pattern determined by using the DAPI staining, as well as TUNEL assay which shows DNA damage especially in the cells after 4 h of exposure to 1,800-MHz RFR. Our results indicate that the applications of 900- and 1,800-MHz (2 W/kg) RFR cause to decrease in the proliferation of the Hep G2 cells after 4 h of exposure. Further studies will be conducted on other frequency bands of RFR and longer duration of exposure. PMID:24817642

  7. Cytotoxic effect of levoglucosenone and related derivatives against human hepatocarcinoma cell lines.

    PubMed

    Giri, Germán F; Danielli, Mauro; Marinelli, Raúl A; Spanevello, Rolando A

    2016-08-15

    Levoglucosenone has been used as template for the synthesis of a wide variety of compounds with an impressive structural variability. However, scarce work has been done regarding the generation of new bioactive entities. Here we report the cytotoxic effect of levoglucosenone and some related derivatives against hepatocarcinoma cell lines. Compounds were obtained in only one synthetic step and one of them showed an activity within the range of IC50 values of cisplatin, a frequently administered chemotherapy drug. PMID:27422336

  8. Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells.

    PubMed

    Michaud, Mickaël; Barakat, Stéphane; Magnard, Sandrine; Rigal, Dominique; Baggetto, Loris G

    2011-01-01

    LRP130 is a ubiquitous protein involved in cellular homeostasis, microtubule alteration, and transactivation of a few multidrug resistance genes. Its role in resistance to apoptosis in HepG2 and HUH7 hepatocarcinoma cells was investigated. Using shRNA-producing lentiviruses to down-regulate the LRP130 gene, we showed that i) LRP130 did not affect the capacity of hepatocarcinoma cells to extrude drugs since LRP130 down-regulation was insufficient to significantly reduce P-glycoprotein production in these cells, and ii) the expression of 11 apoptosis-related genes measured by PCR-array was significantly reduced. Interestingly, six of these genes encode extrinsic pathway proapoptotic proteins whose expression was higher in LRP130-non producing than in LRP130-producing HepG2 cells. Fluorescence microscopy confirmed this new anti-apoptotic role of LRP130, which is strengthened by a significantly reduced cytochrome c oxidase activity in LRP130-down-regulated hepatocarcinoma cells. PMID:21109938

  9. Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells

    PubMed Central

    Adjibade, Pauline; St-Sauveur, Valérie Grenier; Huberdeau, Miguel Quevillon; Fournier, Marie-Josée; Savard, Andreanne; Coudert, Laetitia; Khandjian, Edouard W.; Mazroui, Rachid

    2015-01-01

    Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2α. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2α kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2α-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2α where SGs dampen the activation of the phospho-eIF2α-downstream ATF4 cell death pathway. PMID:26556863

  10. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    SciTech Connect

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing . E-mail: jnzhang@dlmedu.edu.cn

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  11. Multicarotenoids at Physiological Levels Inhibit Metastasis in Human Hepatocarcinoma SK-Hep-1 Cells.

    PubMed

    Chen, Huei-Yan; Yang, Chih-Min; Chen, Jen-Yin; Yueh, Te-Cheng; Hu, Miao-Lin

    2015-01-01

    Several studies have demonstrated that single carotenoid, including lycopene, β-carotene, and α-carotene, exhibits antimetastatic effects; however, little is known whether multicarotenoids have similar effects. Herein, we investigated the antimetastatic effect of multicarotenoids at physiological serum levels in Taiwanese (MCT at 1.4 μM) and American (MCA at 1.8 μM) populations using human hepatocarcinoma SK-Hep-1 cells in comparison with single carotenoid, such as lycopene (0.3 or 0.6 μM, respectively), α-carotene (0.1 μM), β-carotene (0.4 μM), lutein (0.4 or 0.5 μM, respectively), and β-cryptoxanthin (0.2 μM). Results reveal that MCA treatment exhibited an additive inhibition on invasion, migration and adhesion at 24 and 48 h of incubation, whereas MCT treatment possessed additive inhibition at 48 h of incubation. The antimetastatic action of MCT and MCA involved additive reduction on activities of matrix metalloproteinase (MMP)-2, -9, and protein expression of Rho and Rac 1 but additive promotion on protein expression of tissue inhibitor of MMP (TIMP)-1 and -2. All of these effects were stronger in MCA than in MCT at 24 and 48 h of incubation. These results demonstrate that multi-carotenoids effectively inhibit metastasis of human hepatocarcinoma SK-Hep-1 cells. More in vivo studies are needed to confirm these findings. PMID:25868689

  12. Spheroid Formation of Hepatocarcinoma Cells in Microwells: Experiments and Monte Carlo Simulations.

    PubMed

    Wang, Yan; Kim, Myung Hee; Tabaei, Seyed R; Park, Jae Hyeok; Na, Kyuhwan; Chung, Seok; Zhdanov, Vladimir P; Cho, Nam-Joon

    2016-01-01

    The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells. PMID:27571565

  13. Spheroid Formation of Hepatocarcinoma Cells in Microwells: Experiments and Monte Carlo Simulations

    PubMed Central

    Tabaei, Seyed R.; Park, Jae Hyeok; Na, Kyuhwan; Chung, Seok; Zhdanov, Vladimir P.

    2016-01-01

    The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells. PMID:27571565

  14. Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

    PubMed

    Sorimachi, K; Robbins, J

    1979-01-01

    Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation. PMID:107104

  15. Dendrosomal curcumin nanoformulation modulate apoptosis-related genes and protein expression in hepatocarcinoma cell lines.

    PubMed

    Montazeri, Maryam; Sadeghizadeh, Majid; Pilehvar-Soltanahmadi, Yones; Zarghami, Faraz; Khodi, Samaneh; Mohaghegh, Mina; Sadeghzadeh, Hadi; Zarghami, Nosratollah

    2016-07-25

    The side-effects observed in conventional therapies have made them unpromising in curing Hepatocellular carcinoma; therefore, developing novel treatments can be an overwhelming significance. One of such novel agents is curcumin which can induce apoptosis in various cancerous cells, however, its poor solubility is restricted its application. To overcome this issue, this paper employed dendrosomal curcumin (DNC) was employed to in prevent hepatocarcinoma in both RNA and protein levels. Hepatocarcinoma cells, p53 wild-type HepG2 and p53 mutant Huh7, were treated with DNC and investigated for toxicity study using MTT assay. Cell cycle distribution and apoptosis were analyzed using Flow-cytometry and Annexin-V-FLUOS/PI staining. Real-time PCR and Western blot were employed to analyze p53, BAX, Bcl-2, p21 and Noxa in DNC-treated cells. DNC inhibited the growth in the form of time-dependent manner, while the carrier alone was not toxic to the cell. Flow-cytometry data showed the constant concentration of 20μM DNC during the time significantly increases cell population in SubG1 phase. Annexin-V-PI test showed curcumin-induced apoptosis was enhanced in Huh7 as well as HepG2, compared to untreated cells. Followed by treatment, mRNA expression of p21, BAX, and Noxa increased, while the expression of Bcl-2 decreased, and unlike HepG2, Huh7 showed down-regulation of p53. In summary, DNC-treated hepatocellular carcinoma cells undergo apoptosis by changing the expression of genes involved in the apoptosis and proliferation processes. These findings suggest that DNC, as a plant-originated therapeutic agent, could be applied in cancer treatment. PMID:27234697

  16. A novel cell cycle blocker extracted from Stellera chamaejasme L. inhibits the proliferation of hepatocarcinoma cells.

    PubMed

    Kan, Xiao-Xi; Li, Qi; Chen, Xi; Wang, Ya-Jie; Li, Yu-Jie; Yang, Qing; Xiao, Hong-Bin; Wang, Zhi-Xin; Chen, Ying; Weng, Xiao-Gang; Cai, Wei-Yan; Zhu, Xiao-Xin

    2016-06-01

    Currently, liver cancer is the sixth most prevalent cancer and the third most common cause of cancer-related death. However, effective chemotherapeutic drugs with low drug resistance and few side-effects for the clinical treatment of liver cancer are lacking. Therefore, the search for novel drugs to compensate for the defects of existing drugs is urgently needed. Herein, we successfully screened an extract named from Stellera chamaejasme L. (SCL), a historically confimed antitumor plant, through a novel extraction platform. In the present study, we firstly screened the anticancer effect of ESC by the sulforhodamine B (SRB) cell proliferation assay in a wide range of malignant cell lines, including A549, NCI-H157, NCI-H460, SK-HEP-1 and HepG2. With the highest inhibitory rate in hepatocarcinoma cells, we further identified the tumor-suppressive efficacy and the safety of ESC in an H22 hepatocarcinoma xenograft model in vivo. In a mechanistic study, flow cytometry and western blot analysis were performed to evaluate the effects of ESC on the induction of cell apoptosis, intervention of cell cycle distribution and its influence on key G2/M-phase regulators. The results showed that ESC significantly inhibited the cell growth of liver cancer cell lines. Accordingly, the tumor inhibition rate was also increased following ESC administration with little systemic toxicity in H22-transplanted mice. Mechanistically, ESC caused obvious G2/M-phase arrest in both the SK-HEP-1 and HepG2 cell lines without cell apoptosis. Furthermore, cyclin B1 was downregulated, while the phosphorylation level of CDK1 was increased in response to ESC treatment. All these data confirmed that ESC possesses potent anti-proliferative efficacy for hepatocarcinoma through the induction of cyclin-mediated cell cycle arrest. Thus, ESC is a promising candidate for hepatocarcinoma treatment in the future. PMID:27109908

  17. Effects of Nanosized Lithium Carbonate Particles on the Functional Activity of Macrophages During Development of Hepatocarcinoma 29.

    PubMed

    Konenkov, V I; Borodin, Yu I; Makarova, O P; Bgatova, N P; Rachkovskaya, L N

    2015-08-01

    The functional activity of macrophages in response to injection of nanosized lithium carbonate particles after initiation of hepatocarcinoma 29 in male CBA mice was evaluated by the production of NO, arginase activity, and absorption of zymosan granules. In intact animals, NO production by peritoneal macrophages increased by 4 times and arginase activity 3.1 times in response to a single injection of nanosized particles into the hip muscle. The level of NO production by macrophages remained high after 4 and 5 injections, while arginase activity returned to normal. The level of phagocytic peritoneal macrophages increased by 1.4 times after 5 injections of the particles. The level of NO production by macrophages gradually increased in animals with hepatocarcinoma developing in the hip muscle: by 1.6 times on day 3, 3.2 times on day 7, and by 2.6 times on day 13 in comparison with the corresponding parameters in intact animals. The increase of NO production by peritoneal macrophages after tumor process initiation was not paralleled by changes in arginase activity and absorption of zymosan granules. The results indicated that injection of nanosized lithium carbonate particles after inoculation of hepatocarcinoma 29 cells in the right hip muscle tissue was inessential for the function of peritoneal macrophages by the studied parameters. PMID:26388569

  18. Anti-hepatocarcinoma effects of berberine nanosuspension against human HepG2 and Huh7 cells as well as H22 tumor bearing mice

    NASA Astrophysics Data System (ADS)

    Wang, Zhi-ping; Wu, Jun-biao; Zhou, Qun; Wang, Yi-fei; Chen, Tongsheng

    2014-09-01

    Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Berberine (Ber), an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Ber nanosuspension (Ber-NS) composed of Ber and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was prepared by high pressure homogenization technique. Both in vitro and in vivo anti-hepatocarcinoma effects of Ber-NS relative to effcacy of bulk Ber were evaluated. The particle size and zeta potential of Ber-NS were 73.1 +/- 3.7 nm and 6.99 +/- 0.17 mV, respectively. Ber-NS exhibited significant inhibitory effects against human HepG2 and Huh7 cells, and the corresponding IC50 values were 8.1 and 4.7 μg/ml (18.3 and 6.5 μg/ml of Ber solution). In vivo studies also showed higher antitumor efficacy, and inhibition rates was 63.7% (41.4 % of Ber solution) at 100 mg/kg intragastric administration in the H22 solid tumor bearing mice. These results suggest that the delivery of Ber as a nanosuspension is a promising approach for treating hepatocarcinoma.

  19. Cytotoxic activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and hepatocarcinoma cells in vitro

    PubMed Central

    2010-01-01

    Background Cholangiocarcinoma is a serious public health in Thailand with increasing incidence and mortality rates. The present study aimed to investigate cytotoxic activities of crude ethanol extracts of a total of 28 plants and 5 recipes used in Thai folklore medicine against human cholangiocarcinoma (CL-6), human laryngeal (Hep-2), and human hepatocarcinoma (HepG2) cell lines in vitro. Methods Cytotoxic activity of the plant extracts against the cancerous cell lines compared with normal cell line (renal epithelial cell: HRE) were assessed using MTT assay. 5-fluorouracil was used as a positive control. The IC50 (concentration that inhibits cell growth by 50%) and the selectivity index (SI) were calculated. Results The extracts from seven plant species (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, Ligusticum sinense, Mimusops elengi) and one folklore recipe (Pra-Sa-Prao-Yhai) exhibited promising activity against the cholangiocarcinoma CL-6 cell line with survival of less than 50% at the concentration of 50 μg/ml. Among these, the extracts from the five plants and one recipe (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, and Pra-Sa-Prao-Yhai recipe) showed potent cytotoxic activity with mean IC50 values of 24.09, 37.36, 34.26, 40.74, 48.23 and 44.12 μg/ml, respectively. All possessed high activity against Hep-2 cell with mean IC50 ranging from 18.93 to 32.40 μg/ml. In contrast, activity against the hepatoma cell HepG2 varied markedly; mean IC50 ranged from 9.67 to 115.47 μg/ml. The only promising extract was from Zingiber officinal (IC50 = 9.67 μg/ml). The sensitivity of all the four cells to 5-FU also varied according to cell types, particularly with CL-6 cell (IC50 = 757 micromolar). The extract from Atractylodes lancea appears to be both the most potent and most selective against cholangiocarcinoma (IC50 = 24.09 μg/ml, SI = 8.6). Conclusions The ethanolic extracts from

  20. Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma

    PubMed Central

    Teng, Hongming; Yang, Yazong; Wei, Hengyun; Liu, Zundong; Liu, Zhichao; Ma, Yanhong; Gao, Zixiang; Hou, Lin; Zou, Xiangyang

    2015-01-01

    Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways. PMID:26047481

  1. Regression of hepatocarcinoma cells using RNA aptamer specific to alpha-fetoprotein

    SciTech Connect

    Lee, Young Ju; Lee, Seong-Wook

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Identification of RNA aptamer specific to AFP with high affinity. Black-Right-Pointing-Pointer Specific induction of HCC proliferation by AFP. Black-Right-Pointing-Pointer Efficient increase in oncogene expression by AFP. Black-Right-Pointing-Pointer Efficient inhibition of AFP-mediated HCC proliferation by the aptamer. Black-Right-Pointing-Pointer Efficient suppression of AFP-induced oncogene expression of by the aptamer. -- Abstract: Alpha-fetoprotein (AFP) is a cancer-associated fetal protein and has long been utilized as a serum fetal defect/tumor marker to monitor distress/disease progression. In addition, AFP is closely associated with the proliferation of hepatocellular carcinoma. Thus, direct targeting of AFP has been recommended for a therapeutic strategy against hepatocellular carcinoma. In this study, we developed and characterized an RNA aptamer that specifically bound to the alpha-fetoprotein using SELEX technology. The aptamer interacted with the AFP with a K{sub D} of {approx}33 nM. Importantly, the identified aptamer specifically and efficiently inhibited the AFP-mediated proliferation of hepatocarcinoma cells in a dose dependent manner. Moreover, the aptamer efficiently down-regulated AFP-induced expression of oncogenes in the cells. These results indicate that an AFP-specific RNA aptamer could be a useful therapeutic and diagnostic agent against AFP-related hepatocellular carcinoma.

  2. MicroRNAs transfer from human macrophages to hepato-carcinoma cells and inhibit proliferation.

    PubMed

    Aucher, Anne; Rudnicka, Dominika; Davis, Daniel M

    2013-12-15

    Recent research has indicated a new mode of intercellular communication facilitated by the movement of RNA between cells. There is evidence that RNA can transfer between cells in a multitude of ways, including in complex with proteins or lipids or in vesicles, including apoptotic bodies and exosomes. However, there remains little understanding of the function of nucleic acid transfer between human cells. In this article, we report that human macrophages transfer microRNAs (miRNAs) to hepato-carcinoma cells (HCCs) in a manner that required intercellular contact and involved gap junctions. Two specific miRNAs transferred efficiently between these cells--miR-142 and miR-223--and both were endogenously expressed in macrophages and not in HCCs. Transfer of these miRNAs influenced posttranscriptional regulation of proteins in HCCs, including decreased expression of reporter proteins and endogenously expressed stathmin-1 and insulin-like growth factor-1 receptor. Importantly, transfer of miRNAs from macrophages functionally inhibited proliferation of these cancerous cells. Thus, these data led us to propose that intercellular transfer of miRNA from immune cells could serve as a new defense against unwanted cell proliferation or tumor growth. PMID:24227773

  3. Evaluation of BCG administered by scarification for immunotherapy of metastatic hepatocarcinoma in the guinea pig.

    PubMed

    Hanna, M G; Peters, L C; Gutterman, J U; Hersh, E M

    1976-05-01

    In inbred guinea pigs, administration of Mycobacterium bovis strain BCG by scarification at a site distant from an excised skin tumor, but in the regional lymph node drainage, was evaluated for its immunotherapeutic effect on the development of lymph node metastases. Scarification was performed after surgical excision of intradermally transplanted syngeneic (line-10) hepatocarcinoma at a time when microscopic foci of tumor cells were present in regional lymph nodes. Various strains of BCG were evaluated for their immunotherapeutic potential: fresh-frozen Phipps, Pasteur, and Tice; and lyophilized Pasteur, Tice, and Connaught. Scarification commenced 3 days after surgical removal of the tumor and continued once a week for 5 weeks. Only lymph nodes from fresh-frozen Phipps- and Pasteur-scarified animals were significantly smaller than those in the control groups. Differences in lymph node weight correlated histologically with less detectable metastases. This cytostatic effect was short lived; eventually, the metastatic tumor growth was not significantly different from that of control animals. No significant differences were observed in mean survival time: All animals died as a result of metastases 3 months after tumor inoculation. These results demonstrated that limited scarification with BCG of certain strains temporarily inhibits the growth and proliferation of metastases in regional lymph nodes after removal of the primary tumor. PMID:186611

  4. Galactosylated pullulan-curcumin conjugate micelles for site specific anticancer activity to hepatocarcinoma cells.

    PubMed

    Sarika, P R; James, Nirmala Rachel; Nishna, N; Anil Kumar, P R; Raj, Deepa K

    2015-09-01

    Galactosylated pullulan-curcumin conjugate (LANH2-Pu Ald-Cur SA) is developed for target specific delivery of curcumin to hepatocarcinoma cells by five step synthetic strategy, which includes oxidation of pullulan (Pu Ald), introduction of amino group to the targeting ligand (LANH2), grafting of the LANH2 to Pu Ald, modification of curcumin (Cur SA) and conjugation of Cur SA to pullulan. Nongalactosylated pullulan-curcumin conjugate (Pu-Cur SA) is also prepared to compare the enhancement in cytotoxicity offered by the targeting group. Both LANH2-Pu Ald-Cur SA and Pu-Cur SA conjugates could self assemble to micelle in water with hydrodynamic diameters of 355±9nm and 363±10nm, respectively. Both conjugates show spherical morphology and enhance stability of curcumin in physiological pH. Compared to Pu-Cur SA, LANH2-Pu Ald-Cur SA exhibits higher toxicity and internalization towards HepG2 cells. This indicates the enhanced uptake of LANH2-Pu Ald-Cur SA conjugate via ASGPR (asialoglycoprotein receptor) mediated endocytosis into HepG2 cells. PMID:26133239

  5. Nanoalumina induces apoptosis by impairing antioxidant enzyme systems in human hepatocarcinoma cells

    PubMed Central

    Alarifi, Saud; Ali, Daoud; Alkahtani, Saad

    2015-01-01

    Alumina nanoparticles (Al2O3NPs) are gradually used in various areas, including nanomedicine, biosensors, and electronics. The current study aimed to explore the DNA damage and cytotoxicity due to Al2O3NPs on human hepatocarcinoma cells (HepG2). The MTT and neutral red uptake assays showed that Al2O3NPs induce significant cell death in a dose- and time-dependent manner. However, Al2O3NPs induced significant intracellular reactive oxygen species production and elevated lipid peroxidation and superoxide dismutase levels in the HepG2 cells. Al2O3NPs 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 Al2O3NPs 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 Al2O3NPs before their industrial application. PMID:26045665

  6. Red Cooperativa de Tejido Humano del NCI

    Cancer.gov

    Bioespecímenes de calidad son un recurso de investigación oncológica. Uno de los programas de bioespecímenes que han operado por más tiempo es la Red Cooperativa de Tejido Humano, ara descubrimientos básicos e investigación inicial de transferencia.

  7. Vacunas contra los virus del papiloma humano

    Cancer.gov

    Una hoja informativa acerca de las vacunas contra los virus del papiloma humano (VPH) para prevenir infecciones con ciertos tipos de VPH, los cuales son la causa principal del cáncer de cuello del útero o cérvix.

  8. Modulation of Hepatocarcinoma Cell Morphology and Activity by Parylene-C Coating on PDMS

    PubMed Central

    Guimard, Denis; Arakawa, Yasuhiko; Sakai, Yasuyuki; Fujii, Teruo

    2010-01-01

    Background The ability to understand and locally control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology as well as tissue engineering research. We present parylene-C (ParC) deposited on polydimethylsiloxane (PDMS) as a new substratum for in vitro advanced cell culture in the case of Human Hepatocarcinoma (HepG2) cells. Principal Findings Our findings establish that the intrinsic properties of ParC-coated PDMS (ParC/PDMS) influence and modulate initial extracellular matrix (ECM; here, type-I collagen) surface architecture, as compared to non-coated PDMS substratum. Morphological changes induced by the presence of ParC on PDMS were shown to directly affect liver cell metabolic activity and the expression of transmembrane receptors implicated in cell adhesion and cell-cell interaction. These changes were characterized by atomic force microscopy (AFM), which elucidated differences in HepG2 cell adhesion, spreading, and reorganization into two- or three-dimensional structures by neosynthesis of ECM components. Local modulation of cell aggregation was successfully performed using ParC/PDMS micropatterns constructed by simple microfabrication. Conclusion/Significance We demonstrated for the first time the modulation of HepG2 cells' behavior in relation to the intrinsic physical properties of PDMS and ParC, enabling the local modulation of cell spreading in a 2D or 3D manner by simple microfabrication techniques. This work will provide promising insights into the development of cell-based platforms that have many applications in the field of in vitro liver tissue engineering, pharmacology and therapeutics. PMID:20300511

  9. Innate immunity and hepatocarcinoma: Can toll-like receptors open the door to oncogenesis?

    PubMed Central

    Lopes, Jorge André Gomes; Borges-Canha, Marta; Pimentel-Nunes, Pedro

    2016-01-01

    Hepatocarcinoma (HCC) is a highly prevalent cancer worldwide and its inflammatory background was established long ago. Recent studies have shown that innate immunity is closely related to the HCC carcinogenesis. An effective innate immunity response relies on the toll-like receptors (TLR) found in several different liver cells which, through different ligands and many signaling pathways can elicit, not only a pro-inflammatory but also an oncogenic or anti-oncogenic response. Our aim was to study the role of TLRs in the liver oncogenesis and as a consequence their value as potential therapeutic targets. We performed a systematic review of PubMed searching for original articles studying the relationship between HCC and TLRs until March 2015. TLR2 appears to be a fundamental stress-sensor as its absence reveals an augmented tendency to accumulate DNA-damages and to cell survival. However, pathways are still not fully understood as TLR2 up-regulation was also associated to enhanced tumorigenesis. TLR3 has a well-known protective role influencing crucial processes like angiogenesis, cell growth or proliferation. TLR4 works as an interesting epithelial-mesenchymal transition’s inducer and a promoter of cell survival probably inducing HCC carcinogenesis even though an anti-cancer role has already been observed. TLR9’s influence on carcinogenesis is also controversial and despite a potential anti-cancer capacity, a pro-tumorigenic role is more likely. Genetic polymorphisms in some TLRs have been found and its influence on the risk of HCC has been reported. As therapeutic targets, TLRs are already in use and have a great potential. In conclusion, TLRs have been shown to be an interesting influence on the HCC’s microenvironment, with TLR3 clearly determining an anti-tumour influence. TLR4 and TLR9 are considered to have a positive relationship with tumour development even though, in each of them anti-tumorigenic signals have been described. TLR2 presents a more

  10. Global Assessment of Antrodia cinnamomea-Induced MicroRNA Alterations in Hepatocarcinoma Cells

    PubMed Central

    Chan, Yu-Tzu; Huang, Yu-Feng; Ma, Nianhan; Yu, Alice L.; Wu, Chung-Yi; Hu, Miao-Lin; Chiu, Kuo Ping

    2013-01-01

    Recent studies have demonstrated a potent anticancer potential of medicinal fungus Antrodia cinnamomea, especially against hepatocarcinoma. These studies, however, were performed with prolonged treatments, and the early anticancer events remain missing. To probe the early anticancer mechanisms of A. cinnamomea, we treated SK-Hep-1 liver cancer cell with A. cinnamomea fruiting body extract for 2 and 4 hours, sequenced RNA samples with next-generation sequencing approach, and profiled the genome-wide miRNA and mRNA transcriptomes. Results unmistakably associated the early anticancer effect of A. cinnamomea fruiting body extract with a global downregulation of miRNAs which occurred solely in the A. cinnamomea fruiting body extract-treated SK-Hep-1 cells. Moreover, the inhibitory effect of A. cinnamomea fruiting body extract upon cancer miRNAs imposed no discrimination against any particular miRNA species, with oncomirs miR-21, miR-191 and major oncogenic clusters miR-17-92 and miR-106b-25 among the most severely downregulated. Western blotting further indicated a decrease in Drosha and Dicer proteins which play a key role in miRNA biogenesis, together with an increase of XRN2 known to participate in miRNA degradation pathway. Transcriptome profiling followed by GO and pathway analyses indicated that A. cinnamomea induced apoptosis, which was tightly associated with a downregulation of PI3K/AKT and MAPK pathways. Phosphorylation assay further suggested that JNK and c-Jun were closely involved in the apoptotic process. Taken together, our data indicated that the anticancer effect of A. cinnamomea can take place within a few hours by targeting multiple proteins and the miRNA system. A. cinnamomea indiscriminately induced a global downregulation of miRNAs by simultaneously inhibiting the key enzymes involved in miRNA maturation and activating XRN2 protein involved in miRNA degradation. Collapsing of the miRNA system together with downregulation of cell growth and survival

  11. Development of size-customized hepatocarcinoma spheroids as a potential drug testing platform using a sacrificial gelatin microsphere system.

    PubMed

    Leong, Wenyan; Kremer, Antje; Wang, Dong-An

    2016-06-01

    Sacrificial gelatin microspheres can be developed as a cell delivery vehicle for non-anchorage dependent cells - its incorporation into a macroscopic scaffold system not only allows the cells to be cultured in suspension within cavities left behind by the sacrificial material, it also allows scaffold-free tissue development to be confined within the cavities. In this study, dense and highly viable hepatocarcinoma spheroids were developed by means of encapsulation in sacrificial gelatin microspheres produced via a simple water-in-oil emulsion technique. By initial selection of microsphere size and distribution, spheroid size can be controlled for various applications such as uniform tumor spheroids as a reproducible three-dimensional drug screening and testing platform that better mimics the in vivo nature of tumors (instead of conventional monolayer culture), as this study has suggested as a proof-of-concept with chemotherapy drug Doxorubicin. PMID:27040260

  12. Garlic Oil Suppressed Nitrosodiethylamine-Induced Hepatocarcinoma in Rats by Inhibiting PI3K-AKT-NF-κB Pathway.

    PubMed

    Zhang, Cui-Li; Zeng, Tao; Zhao, Xiu-Lan; Xie, Ke-Qin

    2015-01-01

    To explore the underlying mechanisms for the protective effects of garlic oil (GO) against nitrosodiethylamine (NDEA)-induced hepatocarcinoma, 60 male Wistar rats were randomized into 4 groups (n=15): control group, NDEA group, and two GO plus NDEA groups. The rats in GO plus NDEA groups were pretreated with GO (20 or 40 mg/kg) for 7 days. Then, all rats except those in control group were gavaged with NDEA for 20 weeks, and the rats in GO plus NDEA groups were continuously administered with GO. The results showed that GO co-treatment significantly suppressed the NDEA-induced increases of alpha fetal protein (AFP) level in serum, nuclear atypia in H&E staining, sirius red-positive areas and proliferating cell nuclear antigen (PCNA) expression. The molecular mechanisms exploration revealed that the protein levels of phosphatidylinositol 3 kinase (PI3K)-p85, PI3K-p110, total AKT, p-AKT (Ser473) and p-AKT (Thr308) in the liver of NDEA group rats were higher than those in control group rats. In addition, NDEA treatment induced IκB degradation and NF-κB p65 phosphorylation, and up-regulated the protein levels of downstream pro-inflammatory mediators. GO co-treatment significantly reversed all the above adverse effects induced by NDEA. These results suggested that the protective effects of GO against NDEA-induced hepatocarcinoma might be associated with the suppression of PI3K- AKT-NF-κB pathway. PMID:25999787

  13. [V Consensus Meeting of the Spanish Society for Liver Transplant on high-risk recipients, immunosupression scenarios and management of hepatocarcinoma on the transplant waiting list].

    PubMed

    Pardo, Fernando; Pons, José Antonio; Briceño, Javier

    2015-12-01

    With the aim to promote the elaboration of consensus documents on state of the art topics in liver transplantation with multidisciplinary management, the Spanish Society for Liver Transplantation (SETH) organized the V Consensus Meeting with the participation of experts from all the Spanish liver transplant programs. In this edition, the following topics were revised, and we present the summary: 1. High-risk receptors; 2. Immunosuppression scenarios; and 3. Management of the patient with hepatocarcinoma in the waiting list. PMID:26404045

  14. V Consensus Meeting of the Spanish Society for Liver Transplant on high-risk recipients, immunosupression scenarios and management of hepatocarcinoma on the transplant waiting list.

    PubMed

    Pardo, Fernando; Pons, José Antonio; Briceño, Javier

    2015-12-01

    With the aim to promote the elaboration of consensus documents on state of the art topics in liver transplantation with multidisciplinary management, the Spanish Society for Liver Transplantation (SETH) organized the V Consensus Meeting with the participation of experts from all the Spanish liver transplant programs. In this edition, the following topics were revised, and we present the summary: 1. High-risk receptors; 2. Immunosuppression scenarios; and 3. Management of the patient with hepatocarcinoma in the waiting list. PMID:26187597

  15. Effects of melatonin on HIF-1α and VEGF expression and on the invasive properties of hepatocarcinoma cells

    PubMed Central

    COLOMBO, JUCIMARA; MACIEL, JOÃO MARCOS WOLF; FERREIRA, LÍVIA CARVALHO; DA SILVA, RENATO FERREIRA; ZUCCARI, DEBORA APARECIDA PIRES

    2016-01-01

    Liver cancer is the sixth most commonly occurring cancer globally, and the main histological type is hepatocellular carcinoma. This type of neoplasia has a poor prognosis due to a high rate of recurrence and intrahepatic metastasis, which are closely are closely associated with the angiogenic process. Vascular endothelial growth factor (VEGF), which is under the control of hypoxia inducible factor-1α (HIF-1α), stimulates the proliferation of endothelial cells and increases cell permeability, promoting the growth, spread and metastasis of tumors. Melatonin, the main hormone secreted by the pineal gland, may have a significant role in tumor suppression and has demonstrated antiangiogenic and antimetastatic effects. The aim of the present study was to analyze the cell viability, migration and invasion, as well as the expression of proangiogenic proteins VEGF and HIF-1α, in HepG2 hepatocarcinoma cells, following treatment with melatonin. Cells were cultured and cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of proangiogenic proteins VEGF and HIF-1α, under conditions of normoxia and hypoxia, was verified using immunocytochemistry and quantified by densitometry. The analysis of the processes of cell migration and invasion was performed in a Boyden chamber. The MTT assay revealed a reduction in cell viability (P=0.018) following treatment with 1 mM melatonin for 24 h. The expression of proangiogenic proteins VEGF and HIF-1α was reduced in cells treated with 1 mM melatonin for 24 h in normoxic (P<0.001) and hypoxic (P<0.001) conditions, compared with the control group and with induced hypoxia alone. The rate of cell migration and invasion was additionally reduced in cells treated with 1 mM melatonin for 48 h when compared with the control group (P=0.496). The results of the present study suggest that melatonin may have an antiproliferative, antiangiogenic and antimetastatic role in

  16. A novel herbal formulation "LiverCare" differentially regulates primary rat hepatocyte and hepatocarcinoma cell proliferation in vitro.

    PubMed

    Vidyashankar, Satyakumar; Varma, Sandeep R; Azeemudin, Mohammed; Godavarthi, Ashok; Krishna, Nandakumar S; Patki, Pralhad Sadashiv

    2011-09-01

    Hepatocyte growth factor (HGF) plays an important role in hepatocyte proliferation. HGF expression is regulated by various signaling molecules and nuclear receptors. In the present study, LiverCare(®) (LC), a novel polyherbal formulation (The Himalaya Drug Company, Bangalore, India), was evaluated for its efficacy, using co-cultures of primary rat hepatocytes-non-parenchymal cells (NPCs) and human hepatocellular carcinoma cells (HepG2). The rate of primary hepatocyte co-culture proliferation was significantly and dose-dependently increased by LC as determined by [(3)H]thymidine incorporation into newly synthesized DNA and cell proliferation assay. LC also increased HGF expression in primary hepatocyte co-culture. Albumin and urea content remained constant during proliferation of hepatocyte co-cultures in the presence of LC with decreased activity of alanine aminotransferase. It is interesting that LC inhibited incorporation of [(3)H]thymidine into DNA in HepG2 cells. LC enhanced peroxisome proliferator-activated receptor-α expression during hepatocyte proliferation, whereas tumor necrosis factor-α expression remained unaffected. In conclusion, our study clearly showed that LC differentially regulates primary rat hepatocytes and human hepatocarcinoma cell proliferation. LC may be a promising candidate for treating degenerative liver diseases by enhancing liver regeneration. PMID:21812649

  17. Impairment of oxidative phosphorylation increases the toxicity of SYD-1 on hepatocarcinoma cells (HepG2).

    PubMed

    Brandt, Anna Paula; Gozzi, Gustavo Jabor; Pires, Amanda do Rocio Andrade; Martinez, Glaucia Regina; Dos Santos Canuto, André Vinícius; Echevarria, Aurea; Di Pietro, Attilio; Cadena, Sílvia Maria Suter Correia

    2016-08-25

    Toxicity of the SYD-1 mesoionic compound (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) was evaluated on human liver cancer cells (HepG2) grown in either high glucose (HG) or galactose (GAL) medium, and also on suspended cells kept in HG medium. SYD-1 was able to decrease the viability of cultured HepG2 cells in a dose-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays, but no effect was observed on suspended cells after 1-40 min of treatment. Respiration analysis was performed after 2 min (suspended cells) or 24 h (cultured cells) of treatment: no change was observed in suspended cells, whereas SYD-1 inhibited as well basal, leak and uncoupled states of the respiration in cultured cells with HG medium. These inhibitions were consistent with the decrease in pyruvate level and increase in lactate level. Even more extended results were obtained with HepG2 cells grown in GAL medium where, additionally, the ATP amount was reduced. Furthermore, SYD-1 appears not to be transported by the main ABC multidrug transporters. These results show that SYD-1 is able to change the metabolism of HepG2 cells, and suggest that its cytotoxicity is related to impairment of mitochondrial metabolism. Therefore, we may propose that SYD-1 is a potential candidate for hepatocarcinoma treatment. PMID:27417255

  18. Suppression of established hepatocarcinoma in adjuvant only immunotherapy: alum triggers anti-tumor CD8+ T cell response.

    PubMed

    Wang, Bo; Wang, Xuanyi; Wen, Yumei; Fu, Jing; Wang, Hongyang; Ma, Zhangmei; Shi, Yan; Wang, Bin

    2015-01-01

    Dendritic cell-based immunotherapy is a new weapon in our battle against malignancies in human. Recent trials in human and research work in model animals have shown various degrees of success, suggesting its great potential for clinical use. While protocols vary, a common scheme in this category of treatment involves activation of dendritic cells, with the purpose of increasing antigen presentation and cellular immunity. Therefore, proper use of immune adjuvant is a central subject of study. We report here an unexpected finding that injection of alum, the most widely used human adjuvant, into mice carrying H22 hepatocarcinoma resulted in a significant reduction of tumor growth with extended animal survival. This effect was associated with an increased specific CD8(+) T cell activation and an inflammatory environment, yet with minimal overt side effects. Our finding suggests that use of adjuvant alone in certain established tumors can invoke protective host immune activation against the same target, which may be of value in our development of new cancer immunotherapies. PMID:26647964

  19. Suppression of established hepatocarcinoma in adjuvant only immunotherapy: alum triggers anti-tumor CD8+ T cell response

    PubMed Central

    Wang, Bo; Wang, Xuanyi; Wen, Yumei; Fu, Jing; Wang, Hongyang; Ma, Zhangmei; Shi, Yan; Wang, Bin

    2015-01-01

    Dendritic cell-based immunotherapy is a new weapon in our battle against malignancies in human. Recent trials in human and research work in model animals have shown various degrees of success, suggesting its great potential for clinical use. While protocols vary, a common scheme in this category of treatment involves activation of dendritic cells, with the purpose of increasing antigen presentation and cellular immunity. Therefore, proper use of immune adjuvant is a central subject of study. We report here an unexpected finding that injection of alum, the most widely used human adjuvant, into mice carrying H22 hepatocarcinoma resulted in a significant reduction of tumor growth with extended animal survival. This effect was associated with an increased specific CD8+ T cell activation and an inflammatory environment, yet with minimal overt side effects. Our finding suggests that use of adjuvant alone in certain established tumors can invoke protective host immune activation against the same target, which may be of value in our development of new cancer immunotherapies. PMID:26647964

  20. Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

    PubMed

    Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

    2016-02-01

    A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. PMID:26763355

  1. ANXA11 regulates the tumorigenesis, lymph node metastasis and 5-fluorouracil sensitivity of murine hepatocarcinoma Hca-P cells by targeting c-Jun

    PubMed Central

    Wang, Bo; Qi, Houbao; Sun, Ming-Zhong

    2016-01-01

    Annexin A11 (Anxa11) is associated with various cancers. Using a pair of syngeneic murine hepatocarcinoma cells, Hca-P with ~25% and Hca-F with ~75% lymph node metastatic (LNM) potentials, we demonstrated Anxa11 involvement in hepatocarcinoma lymphatic metastasis. Here, ANXA11 acted as a suppressor for the tumorigenicity, LNM and 5-FU resistance of Hca-P via c-Jun. We constructed monoclonal Hca-P cell line with stable ANXA11 knockdown. Although Bax and Bcl-2 levels increased in shRNA-Anxa11-transfected Hca-P, ANXA11 downregulation showed no clear effect on Hca-P apoptosis. ANXA11 downregulation promoted in vitro migration and invasion capacities of Hca-P. In situ adhesion potential of Hca-P cells toward LN was significantly enhanced following ANXA11 downregulation. Consistently, ANXA11 downregulation promoted the in vivo tumor growth and LNM capacities of Hca-P cells. ANXA11 knockdown enhanced the chemoresistance of Hca-P cells specifically toward 5-FU instead of cisplatin. Its downregulation increased c-Jun (pSer73) and decreased c-Jun (pSer243) levels in Hca-P. c-Jun (pSer243) downregulation seemed to be only correlated with ANXA11 knockdown without the connection to 5-FU treatment. Interestingly, compared with scramble-Hca-P cells, the levels of c-Jun and c-Jun (pSer73) in shRNA-Anxa11-Hca-P cells were upregulated in the presences of 0.1 and 1.0 mg/L 5-FU. The levels changes from c-Jun and c-Jun (pSer73) in Hca-P cells showed a more obvious tendency with the combination of ANXA11 knockdown and 5-FU treatment. ANXA11 level regulates LNM and 5-FU resistance of Hca-P via c-Jun pathway. It might play an important role in hepatocarcinoma cell malignancy and be a therapeutic target for hepatocarcinoma. PMID:26908448

  2. Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2) cell line

    PubMed Central

    2011-01-01

    Background Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2) as compared to normal African green monkey kidney epithelial cell lines. Methods Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl) pyridine assay. Results The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6) and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 μg/ml (mean ± standard deviation). Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 μg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 μg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. Conclusion The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells. PMID:22041055

  3. Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents.

    PubMed

    Kita, Kayoko; Suzuki, Toshihide; Ochi, Takafumi

    2007-05-01

    In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments. PMID:17321558

  4. Utilization of metabonomics to identify serum biomarkers in murine H22 hepatocarcinoma and deduce antitumor mechanism of Rhizoma Paridis saponins.

    PubMed

    Qiu, Peiyu; Man, Shuli; Yang, He; Fan, Wei; Yu, Peng; Gao, Wenyuan

    2016-08-25

    Murine H22 hepatocarcinoma model is so popular to be used for the preclinical anticancer candidate's evaluation. However, the metabolic biomarkers of this model were not identified. Meanwhile, Rhizoma Paridis saponins (RPS) as natural products have been found to show strong antitumor activity, while its anti-cancer mechanism is not clear. To search for potential metabolite biomarkers of this model, serum metabonomics approach was applied to detect the variation of metabolite biomarkers and the related metabolism genes and signaling pathway were used to deduce the antitumor mechanisms of RPS. As a result, ten serum metabolites were identified in twenty-four mice including healthy mice, non-treated cancer mice, RPS-treated cancer mice and RPS-treated healthy mice. RPS significantly decreased tumor weight correlates to down-regulating lactate, acetate, N-acetyl amino acid and glutamine signals (p < 0.05), which were marked metabolites screened according to the very important person (VIP), loading plot and receiver operating characteristic curve (ROC) tests. For the analysis of metabolic enzyme related genes, RPS reversed the aerobic glycolysis through activating tumor suppressor p53 and PTEN, and suppressed FASN to inhibit lipogenesis. What's more, RPS repressed Myc and GLS expression and decreased glutamine level. The regulating PI3K/Akt/mTOR and HIF-1α/Myc/Ras networks also participated in these metabolic changes. Taken together, RPS suppressed ATP product made the tumor growth slow, which indicated a good anti-cancer effect and new angle for understanding the mechanism of RPS. In conclusion, this study demonstrated that the utility of (1)H NMR metabolic profiles taken together with tumor weight and viscera index was a promising screening tool for evaluating the antitumor effect of candidates. In addition, RPS was a potent anticancer agent through inhibiting cancer cellular metabolism to suppress proliferation in hepatoma H22 tumor murine, which promoted the

  5. Down-regulation of STAT3 expression using vector-based RNA interference promotes apoptosis in Hepatocarcinoma cells.

    PubMed

    Zhang, Junwei; Du, Jiajun; Liu, Qi; Zhang, Yi

    2016-08-01

    In this study, we followed a DNA vector-based RNAi approach to silence the signal transducer and activator of transcription 3 (STAT3) expression in Bel-7402 cells, to explore how the Janus kinase (JAK)/STAT3 signaling pathway influences the apoptosis of hepatocarcinoma cells. According to GenBank's STAT3 cDNA, the plasmid pGCsi.U6/neoRFP STAT3, which was designed for expression of STAT3 small interfering RNA (siRNA), was constructed and synthesized, and then transfected into Bel-7402 cells using Lipofectamine 2000. Cells with or without siRNA transfection were treated in wells. The apoptotic rate was detected by flow cytometry (FCM) and by staining with the Annexin V/propidium iodide (PI) apoptosis detection kit. Simultaneously, the mitochondrial membrane potential (ΔΨm) was visualized by JC-1 fluorescence staining and observed using the inverted fluorescence microscope. Furthermore, the expression of caspase-3 protein was analyzed by Western blotting. The results showed that treatment with STAT3 siRNA displayed effects in the Bel-7402 cells, causing a significantly increased apoptotic ratio (P < 0.05). The mitochondrial membrane potential of the STAT3 siRNA group, observed by the JC-1 fluorescence staining, decreased significantly. The protein expression of active caspase-3 increased with STAT3 siRNA treatment, and was significantly higher than that of the control group (P < 0.05). STAT3 gene-silencing significantly improves the apoptotic effect against Bel-7402 cells. PMID:26134753

  6. High expression of hexokinase domain containing 1 is associated with poor prognosis and aggressive phenotype in hepatocarcinoma.

    PubMed

    Zhang, Zijian; Huang, Shanzhou; Wang, Huanyu; Wu, Jian; Chen, Dong; Peng, Baogang; Zhou, Qi

    2016-06-10

    Rapid progress and metastasis remain the major treatment failure modes of hepatocarcinoma (HCC). Unfortunately, the underlying molecular mechanisms of hepatoma cell proliferation and migration are poorly understood. Metabolic abnormalities play critical roles in tumorigenesis and progression. Hexokinase domain containing 1 (HKDC1) catalyzes the phosphorylation of glucose. However, the functions and mechanisms of HKDC1 in cancer remain unknown. In this study, real-time RT-PCR and Western blotting assays were used to detect the HKDC1 expression levels in HCC tissues and cell lines. The Oncomine™ Cancer Microarray Database was applied to analysis the correlations between HKDC1 expression and HCC clinical characteristics. MTT and Transwell migration assays were performed to determine the functions of HKDC1 in HCC cells. The effect of HKDC1 on Wnt/β-catenin signaling pathway was assessed using Western blotting assay. In this study, we found that HKDC1 expression levels were elevated in HCC tissues compared with the adjacent tissues. HCC patients with high expression levels of HKDC1 had poor overall survival (OS). Furthermore, higher HKDC1 levels also predicted a worse OS of patients within solitary, elevated pre-operated serum alpha fetoprotein (AFP) level and higher tumor diameter. Moreover, silencing HKDC1 suppressed HCC cells proliferation and migration in vitro. Downregulated HKDC1 expression repressed β-Catenin and c-Myc expression, which indicates that silencing HKDC1 may reduce proliferation and migration via inhibiting the Wnt/β-catenin signaling pathway in HCC. In summary, HKDC1 provides further insight into HCC tumor progression and may provide a novel prognostic biomarker and therapeutic target for HCC treatment. PMID:27155152

  7. Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents

    SciTech Connect

    Kita, Kayoko . E-mail: kkayoko@pharm.teikyo-u.ac.jp; Suzuki, Toshihide; Ochi, Takafumi

    2007-05-01

    In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments.

  8. Ginseng (Panax quinquefolius) and Licorice (Glycyrrhiza uralensis) Root Extract Combinations Increase Hepatocarcinoma Cell (Hep-G2) Viability

    PubMed Central

    Popovich, David G.; Yeo, Shi Yun; Zhang, Wei

    2011-01-01

    The combined cytoactive effects of American ginseng (Panax quinquefolius) and licorice (Glycyrrhiza uralensis) root extracts were investigated in a hepatocarcinoma cell line (Hep-G2). An isobolographic analysis was utilized to express the possibility of synergistic, additive or antagonistic interaction between the two extracts. Both ginseng and licorice roots are widely utilized in traditional Chinese medicine preparations to treat a variety of ailments. However, the effect of the herbs in combination is currently unknown in cultured Hep-G2 cells. Ginseng (GE) and licorice (LE) extracts were both able to reduce cell viability. The LC50 values, after 72 h, were found to be 0.64 ± 0.02 mg/mL (GE) and 0.53 ± 0.02 mg/mL (LE). An isobologram was plotted, which included five theoretical LC50s calculated, based on the fixed fraction method of combination ginseng to licorice extracts to establish a line of additivity. All combinations of GE to LE (1/5, 1/3, 1/2, 2/3, 4/5) produced an effect on Hep-G2 cell viability but they were all found to be antagonistic. The LC50 of fractions 1/3, 1/2, 2/3 were 23%, 21% and 18% above the theoretical LC50. Lactate dehydrogenase release indicated that as the proportion of GE to LE increased beyond 50%, the influence on membrane permeability increased. Cell-cycle analysis showed a slight but significant arrest at the G1 phase of cell cycle for LE. Both GE and LE reduced Hep-G2 viability independently; however, the combinations of both extracts were found to have an antagonistic effect on cell viability and increased cultured Hep-G2 survival. PMID:19617200

  9. Gelsolin negatively regulates the activity of tumor suppressor p53 through their physical interaction in hepatocarcinoma HepG2 cells

    SciTech Connect

    An, Joo-Hee; Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; Kang, Eun-Jin; Choi, Kyung-Hee

    2011-08-19

    Highlights: {yields} The actin binding protein Gelsolin (GSN) interacts with transcription factor p53. {yields} GSN interacts with transactivation- and DNA binding domains of p53. {yields} GSN represses transactivity of p53 via inhibition of nuclear translocation of p53. {yields} GSN inhibits the p53-mediated apoptosis in hepatocarcinoma HepG2 cells. -- Abstract: As a transcription factor, p53 modulates several cellular responses including cell-cycle control, apoptosis, and differentiation. In this study, we have shown that an actin regulatory protein, gelsolin (GSN), can physically interact with p53. The nuclear localization of p53 is inhibited by GSN overexpression in hepatocarcinoma HepG2 cells. Additionally, we demonstrate that GSN negatively regulates p53-dependent transcriptional activity of a reporter construct, driven by the p21-promoter. Furthermore, p53-mediated apoptosis was repressed in GSN-transfected HepG2 cells. Taken together, these results suggest that GSN binds to p53 and this interaction leads to the inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm in HepG2 cells.

  10. In vitro and in vivo magnetic resonance imaging with chlorotoxin-conjugated superparamagnetic nanoprobes for targeting hepatocarcinoma.

    PubMed

    Chen, Zhu; Xiao, En-Hua; Kang, Zhen; Zeng, Wen-Bin; Tan, Hui-Long; Li, Hua-Bing; Bian, Du-Jun; Shang, Quan-Liang

    2016-05-01

    The present study aimed to assess the in vitro and in vivo magnetic resonance imaging (MRI) features of chlorotoxin (CTX)-conjugated superparamagnetic iron oxide (SPIO) nanoprobes. CTX-conjugated nanoprobes were composed of SPIO coated with polyethylene glycol (PEG) and conjugated with CTX. The nanoprobes were termed SPIO-PEG-CTX. MRI of the SPIO and SPIO-PEG-CTX solutions at a different concentration was performed with a 3.0-T MRI scanner (Philips Achieva 3.0T X Series; Phillips Healthcare, The Netherlands). Rabbit VX2 hepatocarcinoma was established by a traditional laparotomy method (injection of the tumor particles into the liver using a 15G syringe needle) following approval by the institutional animal care and use committee. Contrast-enhanced MRI of VX2 rabbits (n=8) was performed using the same MRI scanner with SPIO‑PEG-CTX solutions as the contrast agent. Data were analyzed with calibration curve and a paired t-test. The SPIO-PEG-CTX nanoparticles were successfully prepared. With increasing concentrations of the solutions, the MRI signal intensity was increased at T1WI, but decreased at T2WI, which were the same as that for SPIO. Rabbit VX2 carcinoma appeared as a low MRI signal at T1WI, and high at T2WI. After injection of the contrast agent, the MRI signal of carcinoma was decreased relative to that before injection at T2WI (1,161±331.5 vs. 1,346±300.5; P=0.004<0.05), while the signal of the adjacent normal hepatic tissues was unchanged (480.6±165.1 vs. 563.4±67.8; P=0.202>0.05). The SPIO-PEG-CTX nanoparticles showed MRI negative enhancement at T2WI and a targeting effect in liver cancer, which provides the theoretical basis for further study of the early diagnosis of hepatocellular carcinoma. PMID:26934940

  11. (24S)-ergostane-3β,5α,6β-triol from Hedyotis chrysotricha with inhibitory activity on migration of SK-HEP-1 human hepatocarcinoma cells.

    PubMed

    Ye, Miao; Su, Jing-Jing; Liu, Shu-Ting; Cao, Lei; Xiong, Juan; Zhao, Yun; Fan, Hui; Yang, Guo-Xun; Xia, Gang; Hu, Jin-Feng

    2013-01-01

    The methanol extract of the whole plant of Hedyotis chrysotricha demonstrated cytotoxicity against SK-HEP-1 human hepatocarcinoma cells in a primary screening for novel antitumour agents. Bioassay-guided fractionation and purification led to an active principle (24S)-ergostane-3β,5α,6β-triol (1) along with four inactive compounds (2-5). The in vitro transwell migration assay showed that compound 1 remarkably reduced the migration of SK-HEP-1 cells by 78.9% at a dose of 30 µM, without any apoptotic effect on this cell line. Moreover, all the isolated compounds were further evaluated for their cytotoxicities against another four human cancer cell lines (MCF-7, NUGC3, SH-SY5Y and PC-3). PMID:22889249

  12. Localization of /sup 111/In- and /sup 125/I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors

    SciTech Connect

    Bernhard, M.I.; Hwang, K.M.; Foon, K.A.; Keenan, A.M.; Kessler, R.M.; Frincke, J.M.; Tallam, D.J.; Hanna, M.G. Jr.; Peters, L.; Oldham, R.K.

    1983-09-01

    A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either /sup 125/I or /sup 111/In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

  13. Application of liver three-dimensional printing in hepatectomy for complex massive hepatocarcinoma with rare variations of portal vein: preliminary experience

    PubMed Central

    Xiang, Nan; Fang, Chihua; Fan, Yingfang; Yang, Jian; Zeng, Ning; Liu, Jun; Zhu, Wen

    2015-01-01

    Background: To discuss the role of Liver 3D printing in the treatment of complex massive hepatocarcinoma with rare variations of portal vein. Methods: Data of enhanced computed tomography (CT) were imported into the medical image three-dimensional visualization system (MI-3DVS) to create Standard Template Library (STL) files, which were read by 3D printer to construct life-size 3D physical liver model. The preoperative surgical planning was performed on the 3D model according to individualized segmentation, volume calculation, and virtual operation. Results: The 3D printing liver model was consistent with the model in MI-3DVS. The segment 4 portal vein (S4PV) was absent and variant S4PV originated from right anterior portal vein (RAPV). The preoperative surgical planning was designed according to the relationship between tumor and portal vein variation. Theoretically, the residual liver volume was 40.76%, if the right hemihepatctomy was carried out after the trunk of right portal vein (RPV) ligated. However, the actual residual volume was only 21.37% due to the variant S4PV originates from RAPV, thus, right trisegmentectomy would have to be performed. Interestingly, after optimization, the residual liver volume increased to 57.25% as narrow-margin right hemihepatectomy with the variant S4PV reserved were performed. The final resection was determined to be narrow-margin right hemihepatectomy. The actual surgical procedure was consistent with the preoperative surgical planning. Conclusion: Liver 3D printing may be a safe and effective technique to improve the success rate of surgery and reduce the operation risk for patients with complex massive hepatocarcinoma with variations of portal vein. PMID:26770510

  14. Tetrahydro Iso-Alpha Acids and Hexahydro Iso-Alpha Acids from Hops Inhibit Proliferation of Human Hepatocarcinoma Cell Lines and Reduce Diethylnitrosamine Induced Liver Tumor Formation in Rats.

    PubMed

    Stärkel, Peter; De Saeger, Christine; Delire, Bénédicte; Magat, Julie; Jordan, Bénédicte; Konda, Veera R; Tripp, Mathew L; Borbath, Ivan

    2015-01-01

    Chronic inflammation plays important role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no antiinflammatory approach has shown its efficacy in preventing HCC occurrence in humans. Because tetra- and hexahydro isoalpha acids (THIAA and HHIAA) from hops elicit antiinflammatory properties, we evaluated these compounds for antitumor effects in vitro in human HCC cell lines (HepG2, Hep3B, Huh7) and in vivo in diethylnitrosamine (DEN)-induced animal model of HCC. In human HCC cell lines, THIAA and HHIAA reduced cell proliferation and viability which was associated with the inhibition of the NF-κB-DNA binding and tumor necrosis factor α mRNA expression. Both compounds also inhibited phosphorylation of the mTOR effector p70S6 kinase without affecting ERK, AKT, JNK, and GSK3β phosphorylation or activator protein-1 activation. In DEN-treated rats, administration of THIAA and HHIAA in food reduced the tumor numbers and the expression of the cellular transformation marker glutathione-S-transferase in the liver. In conclusion, THIAA and HHIAA show antitumor properties in vitro in human HCC cell lines as well as in vivo in a chemically induced animal model of HCC. PMID:25941903

  15. Nimbolide, a neem limonoid abrogates canonical NF-κB and Wnt signaling to induce caspase-dependent apoptosis in human hepatocarcinoma (HepG2) cells.

    PubMed

    Kavitha, Krishnamurthy; Vidya Priyadarsini, Ramamurthi; Anitha, Prabukumar; Ramalingam, Krishnan; Sakthivel, Ramasamy; Purushothaman, Gempuraj; Singh, Abhishek Kumar; Karunagaran, Devarajan; Nagini, Siddavaram

    2012-04-15

    Nuclear factor kappa B (NF-κB), an oncogenic signaling factor plays a critical role in the development and progression of various cancers. The objective of this study was to investigate the effect of nimbolide, a neem derived tetranortriterpenoid on NF-κB signaling and its downstream events - Wnt/β-catenin activation and apoptosis evasion in human hepatocarcinoma (HepG2) cells by evaluating NF-κB family members (NF-κB-p50, p65, IκB-α, p-IκB-α, and IKKβ), members of Wnt signaling (GSK-3β and β-catenin), and intrinsic apoptosis (Bcl-2, Bax, cytochrome c, Smac/DIABLO, caspase-3, and caspase-9). Our results demonstrate that nimbolide concurrently abrogates canonical NF-κB and Wnt signaling and induces intrinsic apoptosis in HepG2 cells. These data suggest that phytochemicals such as nimbolide that can target multiple steps along the NF-κB signaling circuit are promising candidates for future phytochemical-based mechanistic pathway targeted anticancer regimens. PMID:22327045

  16. Turmeric enhancing anti-tumor effect of Rhizoma paridis saponins by influencing their metabolic profiling in tumors of H22 hepatocarcinoma mice.

    PubMed

    Man, Shuli; Chai, Hongyan; Qiu, Peiyu; Liu, Zhen; Fan, Wei; Wang, Jiaming; Gao, Wenyuan

    2015-12-01

    Rhizoma Paridis saponins combined with turmeric (RT) showed well anti-hepatocarcinoma activities in our previous research. The aim of this study was to investigate the progression of the biochemical response to RT and capture metabolic variations during intragastric administration of their compatibility. In the experiment, histopathological examination and (1)H NMR method were developed and validated for the metabolic profiling of RT intervention in H22 tumor growth. Data were analyzed with principal components analysis (PCA) and partial least-squares discrimination analysis (PLS-DA). As a result, Rhizoma paridis saponins (RPS) or RT induced inflammatory cell infiltration in tumors. RT also mediated the tumor microenvironment to promote anti-tumor immunity of mice. RT significantly inhibited tumor growth rate through suppressing levels of amino acids containing alanine, asparagine, glutamine, putrescine, and sarcosine, lipid compounds, and carbohydrates like myo-inositol and arabinose in the tumor tissues. In conclusion, these results uncovered unexpectedly poor nutritional conditions in the RT-treated tumor tissues whose effect was stronger than RPS's. Therefore, RT could be a novel anticancer agent that targets on cancer metabolism through starving tumors reducing viability of cancer cells. PMID:26471217

  17. Vasorin is a potential serum biomarker and drug target of hepatocarcinoma screened by subtractive-EMSA-SELEX to clinic patient serum

    PubMed Central

    Wang, Wei; Huang, Aixue; Li, Jie; Qin, Xingliang; Li, Fei; Lu, Guanyi; Ding, Hongmei; Su, Xueting; Hou, Lvbin; Xia, Wei; Shi, Ming; Zhang, Hongwen; Zhao, Qiang; Dong, Jie; Ge, Xingfeng; Sun, Leqiao; Bai, Chenjun; Wang, Chaonan; Shen, Xuelian; Fang, Tao; Wang, Fusheng; Zhang, Heqiu; Shao, Ningsheng

    2015-01-01

    We report a new biomarker of hepatocarcinoma, vasorin (VASN), screened by a subtractive EMSA-SELEX strategy from AFP negative serum of hepatocellular carcinoma (HCC) patients with extrahepatic metastases. VASN was verified to be highly expressed in sera of 100 cases of HCC patients compared with 97 cases of normal persons and 129 cases of hepatitis patients. Further validation by Q-PCR, IFA and Western blot showed higher expression of VASN at mRNA and protein levels in HCC cell lines and HCC tissues than in normal controls. RNA interference and forced overexpression assays verified that VASN promotes cell proliferation and migration and inhibits apoptosis. Down-regulation of microRNA miR145 and miR146a is an important mechanism leading to high expression of VASN. Conclusion: As a membrane protein and/or as free protein, VASN may be an effective target for biological treatment of liver cancer and is a potential biomarker for HCC diagnosis. Small molecular nucleotides targeting VASN are promising biological therapies to HCC. PMID:25826090

  18. β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells

    PubMed Central

    Gong, Min; Liu, Ying; Zhang, Jian; Gao, Ya-jie; Zhai, Ping-ping; Su, Xi; Li, Xiang; Li, Yan; Hou, Li; Cui, Xiao-nan

    2015-01-01

    Objective. To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. Methods. After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. Results. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. Conclusion. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα. PMID:26221582

  19. POD-1/TCF21 Reduces SHP Expression, Affecting LRH-1 Regulation and Cell Cycle Balance in Adrenocortical and Hepatocarcinoma Tumor Cells

    PubMed Central

    França, Monica Malheiros; Ferraz-de-Souza, Bruno; Lerario, Antonio Marcondes; Fragoso, Maria Candida Barisson Villares; Lotfi, Claudimara Ferini Pacicco

    2015-01-01

    POD-1/TCF21 may play a crucial role in adrenal and gonadal homeostasis and represses Sf-1/SF-1 expression in adrenocortical tumor cells. SF-1 and LRH-1 are members of the Fzt-F1 subfamily of nuclear receptors. LRH-1 is involved in several biological processes, and both LRH-1 and its repressor SHP are involved in many types of cancer. In order to assess whether POD-1 can regulate LRH-1 via the same mechanism that regulates SF-1, we analyzed the endogenous mRNA levels of POD-1, SHP, and LRH-1 in hepatocarcinoma and adrenocortical tumor cells using qRT-PCR. Hereafter, these tumor cells were transiently transfected with pCMVMycPod-1, and the effect of POD-1 overexpression on E-box elements in the LRH-1 and SHP promoter region were analyzed by ChIP assay. Also, Cyclin E1 protein expression was analyzed to detect cell cycle progression. We found that POD-1 overexpression significantly decreased SHP/SHP mRNA and protein levels through POD-1 binding to the E-box sequence in the SHP promoter. Decreased SHP expression affected LRH-1 regulation and increased Cyclin E1. These findings show that POD-1/TCF21 regulates SF-1 and LRH-1 by distinct mechanisms, contributing to the understanding of POD-1 involvement and its mechanisms of action in adrenal and liver tumorigenesis, which could lead to the discovery of relevant biomarkers. PMID:26421305

  20. Viscum Album Var Hot Water Extract Mediates Anti-cancer Effects through G1 Phase Cell Cycle Arrest in SK-Hep1 Human Hepatocarcinoma cells.

    PubMed

    dela Cruz, Joseph Flores; Kim, Yeon Soo; Lumbera, Wenchie Marie Lara; Hwang, Seong Gu

    2015-01-01

    Viscum album var (VAV) also known as mistletoe, has long been categorized as a traditional herbal medicine in Asia. In addition to its immunomodulating activities, mistletoe has also been used in the treatment of chronic hepatic disorders in China and Korea. There are numerous reports showing that VAV possesses anti-cancer effects, however influence on human hepatocarcinoma has never been elucidated. In the present study, hot water extracts of VAV was evaluated for its potential anti-cancer effect in vitro. SK-Hep1 cells were treated with VAV (50-400 ug/ml) for both 24 and 48 hours then cell viability was measured by cell counting kit-8 (CCK-8). Flow cytometry analysis was used to measure the proportion of SK-Hep1 in the different stages of cell cycle. RT-PCR and Western blot analysis were conducted to measure expression of cell cycle arrest related genes and proteins respectively. VAV dose dependently inhibited the proliferation of SK-Hep1 cells without any cytotoxicity with normal Chang liver cell (CCL-13). Flow cytometry analysis showed that VAV extract inhibited the cell cycle of SK-Hep1 cells via G1 phase arrest. RT-PCR and Western blot analysis both revealed that cyclin dependent kinase 2 (Cdk2) and cyclin D1 gene expression were significantly down regulated while p21 was upregulated dose dependently by VAV treatment. Combined down regulation of Cdk2, Cyclin D1 and up regulation of p21 can result in cell death. These results indicate that VAV showed evidence of anti-cancer activity through G1 phase cell cycle arrest in SK-Hep1 cells. PMID:26434853

  1. Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening.

    PubMed

    Wilson, Jodie; Berntsen, Hanne Friis; Zimmer, Karin Elisabeth; Frizzell, Caroline; Verhaegen, Steven; Ropstad, Erik; Connolly, Lisa

    2016-03-01

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POPs were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p'-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2h and 48h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC+Br mixture in comparison to the effects of the individual mixtures. No significant effects were detected in the Br+Cl, PFC+Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment. PMID:26772051

  2. MiR-338-3p Inhibits Hepatocarcinoma Cells and Sensitizes These Cells to Sorafenib by Targeting Hypoxia-Induced Factor 1α

    PubMed Central

    Fang, Qiuju; Sun, Jianmin; Zhang, Songyan; Zhan, Chao; Liu, Shujie; Zhang, Yubao

    2014-01-01

    Hypoxia is a common feature of solid tumors and an important contributor to anti-tumor drug resistance. Hypoxia inducible factor-1 (HIF-1) is one of the key mediators of the hypoxia signaling pathway, and was recently proven to be required for sorafenib resistance in hepatocarcinoma (HCC). MicroRNAs have emerged as important posttranslational regulators in HCC. It was reported that miR-338-3p levels are associated with clinical aggressiveness of HCC. However, the roles of miR-338-3p in HCC disease and resistance to its therapeutic drugs are unknown. In this study, we found that miR-338-3p was frequently down-regulated in 14 HCC clinical samples and five cell lines. Overexpression of miR-338-3p inhibited HIF-1α 3′-UTR luciferase activity and HIF-1α protein levels in HepG2, SMMC-7721, and Huh7 cells. miR-338-3p significantly reduced cell viability and induced cell apoptosis of HCC cells. Additionally, HIF-1α overexpression rescued and HIF-1α knock-down abrogated the anti-HCC activity of miR-338-3p. Furthermore, miR-338-3p sensitized HCC cells to sorafenib in vitro and in a HCC subcutaneous nude mice tumor model by inhibiting HIF-1α. Collectively, miR-338-3p inhibits HCC tumor growth and sensitizes HCC cells to sorafenib by down-regulating HIF-1α. Our data indicate that miR-338-3p could be a potential candidate for HCC therapeutics. PMID:25531114

  3. Can macroalgae provide promising anti-tumoral compounds? A closer look at Cystoseira tamariscifolia as a source for antioxidant and anti-hepatocarcinoma compounds

    PubMed Central

    Vizetto-Duarte, Catarina; Custódio, Luísa; Acosta, Gerardo; Lago, João H.G.; Morais, Thiago R.; Bruno de Sousa, Carolina; Gangadhar, Katkam N.; Rodrigues, Maria João; Pereira, Hugo; Lima, Raquel T.; Vasconcelos, M. Helena; Barreira, Luísa; Rauter, Amélia P.; Albericio, Fernando

    2016-01-01

    Marine organisms are a prolific source of drug leads in a variety of therapeutic areas. In the last few years, biomedical, pharmaceutical and nutraceutical industries have shown growing interest in novel compounds from marine organisms, including macroalgae. Cystoseira is a genus of Phaeophyceae (Fucales) macroalgae known to contain bioactive compounds. Organic extracts (hexane, diethyl ether, ethyl acetate and methanol extracts) from three Cystoseira species (C. humilis, C. tamariscifolia and C. usneoides) were evaluated for their total phenolic content, radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, and antiproliferative activity against a human hepatocarcinoma cell line (HepG2 cells). C. tamariscifolia had the highest TPC and RSA. The hexane extract of C. tamariscifolia (CTH) had the highest cytotoxic activity (IC50 = 2.31 µg/mL), and was further tested in four human tumor (cervical adenocarcinoma HeLa; gastric adenocarcinoma AGS; colorectal adenocarcinoma HCT-15; neuroblastoma SH-SY5Y), and two non-tumor (murine bone marrow stroma S17 and human umbilical vein endothelial HUVEC) cell lines in order to determine its selectivity. CTH strongly reduced viability of all tumor cell lines, especially of HepG2 cells. Cytotoxicity was particularly selective for the latter cells with a selectivity index = 12.6 as compared to non-tumor cells. Incubation with CTH led to a 2-fold decrease of HepG2 cell proliferation as shown by the bromodeoxyuridine (BrdU) incorporation assay. CTH-treated HepG2 cells presented also pro-apoptotic features, such as increased Annexin V/propidium iodide (PI) binding and dose-dependent morphological alterations in DAPI-stained cells. Moreover, it had a noticeable disaggregating effect on 3D multicellular tumor spheroids. Demethoxy cystoketal chromane, a derivative of the meroditerpenoid cystoketal, was identified as the active compound in CTH

  4. A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

    PubMed

    Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

    2014-02-28

    NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. PMID:24491563

  5. Antiplatelet drugs induce apoptosis in cultured cancer cells.

    PubMed

    Chen, W H; Yin, H L; Chang, Y Y; Lan, M Y; Hsu, H Y; Liu, J S

    1997-10-01

    In order to understand if antiplatelet drugs possess direct antineoplastic property, we tested the apoptotic effect of 5 popularly marketed antiplatelet drugs in Taiwan in 6 cultured cancer cell lines (Hep 3B hepatocarcinoma, U87-MG malignant glioma, PC-3 prostate adenocarcinoma, HeLa cervical adenocarcinoma, HL-60 preleukemia and K-562 chronic myelogenous leukemia). While acetylsalicylate and flunarizine exerted no effect on these cancer cells, pentoxifyline (PTX), dipyridamole (DYA) and ticlopidine hydrochloride (T. HCl) displayed a time and dose-dependent apoptotic effect on them except for HL-60 and K-562 cells. PTX induced apoptosis in U87-MG, Hep 3B and HeLa cells, DYA in HeLa cells, while T. HCl in U87-MG, Hep 3B, PC-3 and HeLa cells. Adriamycin also provoked apoptotic effect in all 6 cell lines but neither PTX, DYA nor T. HCl acted synergy with adriamycin to HeLa cells, implicating that they may share a similar pathway for inducing apoptosis. Therefore, our results show that the antiplatelet drugs do possess antineoplastic property in vitro. A co-administration of antiplatelet drugs is noteworthy for an alternative adjunctive therapy in cancer patients. PMID:9385774

  6. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

    SciTech Connect

    Liu Hua; Luan Fang; Ju Ying; Shen Hongyu; Gao Lifen; Wang Xiaoyan; Liu Suxia; Zhang Lining; Sun Wensheng; Ma Chunhong . E-mail: machunhong@sdu.edu.cn

    2007-04-06

    The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.

  7. SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells.

    PubMed

    Figarola, James L; Singhal, Jyotsana; Tompkins, Joshua D; Rogers, George W; Warden, Charles; Horne, David; Riggs, Arthur D; Awasthi, Sanjay; Singhal, Sharad S

    2015-12-18

    Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. PMID:26534958

  8. Hydroxysteroid Sulfotransferase SULT2B1b Promotes Hepatocellular Carcinoma Cells Proliferation In Vitro and In Vivo

    PubMed Central

    Yang, Xiaoming; Xu, Yali; Guo, Fenghua; Ning, Yanxia; Zhi, Xiuling; Yin, Lianhua; Li, Xiaobo

    2013-01-01

    Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) is highly selective for the addition of sulfate groups to 3β-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC. PMID:23593328

  9. Production of a bioactive sweetener steviolbioside via specific hydrolyzing ester linkage of stevioside with a β-galactosidase.

    PubMed

    Chen, Jun-ming; Ding, Li; Sui, Xiao-chen; Xia, Yong-mei; Wan, Hui-da; Lu, Tong

    2016-04-01

    A β-galactosidase from Kluyveromyces lactis was found to specifically catalyze hydrolysis of the glycosyl ester linkage of stevioside to yield steviolbioside, a rare sweetener that also exists in Stevia rebaudiana leaves. In a packed bed reactor, a reaction coupling separation was realized and a production yield of steviolbioside reached 90% in 6 h. The hydrolysis product steviolbioside presented higher cytoxicity on human normal cells (hepatocytes cell L02 and intestinal epithelial cell T84) than stevioside did. Comparing to the typical chemotherapy agent, 5-fluorouracil (5-FU), steviolbioside presents much lower cytotoxicity on all assayed human normal cells; it presented notable inhibition on human hepatocarcinoma cell Hep3B, human breast cancer cell MDA-MB-231 and human pancreatic cancer cell BxPC-3. The remarkable inhibition on MDA-MB-231 cells makes steviolbioside a potential remedy for human breast cancer, when steviolbioside is served as a natural sweetener. PMID:26593477

  10. Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

    PubMed

    Dragan, Y; Valdés, R; Gomez-Angelats, M; Felipe, A; Javier Casado, F; Pitot, H; Pastor-Anglada, M

    2000-08-01

    Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity. PMID:10915730

  11. Telomere and telomerase in chronic liver disease and hepatocarcinoma.

    PubMed

    Carulli, Lucia; Anzivino, Claudia

    2014-05-28

    The pathogenesis of liver cirrhosis is not completely elucidated. Although in the majority of patients, the risk factors may be identified in B and C viral hepatitis, alcohol intake, drugs or fatty liver disease, there is a small percentage of patients with no apparent risk factors. In addition, the evolution of chronic liver disease is highly heterogeneous from one patient to another. Among patient with identical risk factors, some rapidly progress to cirrhosis and hepatocellular carcinoma (HCC) whereas others have a benign course. Therefore, a genetic predisposition may contribute to the development of cirrhosis and HCC. Evidence supporting the role of genetic factors as a risk for cirrhosis has been accumulating during the past years. In addition to the results from epidemiological studies, polymorphisms studies and data on twins, the concept of telomere shortening as a genetic risk factor for chronic liver disease and HCC has been proposed. Here we review the literature on telomerase mutations, telomere shortening and liver disease including hepatocellular carcinoma. PMID:24876749

  12. Effect of Ecuador's cash transfer program (Bono de Desarrollo Humano) on child development in infants and toddlers: a randomized effectiveness trial.

    PubMed

    Fernald, Lia C H; Hidrobo, Melissa

    2011-05-01

    We examined the effects of Ecuador's Bono de Desarrollo Humano (BDH)--an unconditional cash transfer program that was rolled-out using a randomized design--on health and development outcomes in very young children. Communities that were randomly assigned to the treatment group began receiving the BDH in 2004 and those randomly assigned to the comparison group began receiving benefits two years later. Families enrolled in the BDH received a monthly cash stipend ($15USD) representing an approximate 6-10% increase in household income. Participants analyzed in this study are children aged 12-35 months from treatment (n = 797) and comparison (n = 399) communities in rural and urban Ecuador. Main outcomes measured were language skills (the Fundación MacArthur Inventorio del Desarollo de Habilidades Comunicativas-Breve), height-for-age z-score, and hemoglobin concentration. Results indicate that in rural areas, being randomized to receive the BDH in very early childhood led to significantly better performance on the number of words a child was saying, and on the probability that the child was combining two or more words. There were no significant effects on language development for children in urban areas and there were no effects on height-for-age z-score or hemoglobin concentration in rural or urban areas. A limited number of potential pathways with respect to cognitive/language stimulation, health behaviors, and parenting quality were also explored. Findings indicate that compared to children in comparison areas, rural children in treatment areas were more likely to have received vitamin A or iron supplementation and have been bought a toy in the past six months. This study provides evidence for significant benefits of an unconditional cash transfer program for language development in very young children in rural areas. PMID:21531060

  13. Down-regulation of EPHX2 gene transcription by Sp1 under high-glucose conditions.

    PubMed

    Oguro, Ami; Oida, Shoko; Imaoka, Susumu

    2015-09-15

    sEH (soluble epoxide hydrolase), which is encoded by the EPHX2 gene, regulates the actions of bioactive lipids, EETs (epoxyeicosatrienoic acids). Previously, we found that high-glucose-induced oxidative stress suppressed sEH levels in a hepatocarcinoma cell line (Hep3B) and sEH was decreased in streptozotocin-induced diabetic mice in vivo. In the present study, we investigated the regulatory mechanisms underlying EPHX2 transcriptional suppression under high-glucose conditions. The decrease in sEH was prevented by an Sp1 (specificity protein 1) inhibitor, mithramycin A, and overexpression or knockdown of Sp1 revealed that Sp1 suppressively regulated sEH expression, in contrast with the general role of Sp1 on transcriptional activation. In addition, we found that AP2α (activating protein 2α) promoted EPHX2 transcription. The nuclear transport of Sp1, but not that of AP2α, was increased under high glucose concomitantly with the decrease in sEH. Within the EPHX2 promoter -56/+32, five Sp1-binding sites were identified, and the mutation of each of these sites showed that the first one (SP1_1) was important in both suppression by Sp1 and activation by AP2α. Furthermore, overexpression of Sp1 diminished the binding of AP2α by DNA-affinity precipitation assay and ChIP, suggesting competition between Sp1 and AP2α on the EPHX2 promoter. These findings provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes. PMID:26341485

  14. Mechanisms regulating cell membrane localization of the chemokine receptor CXCR4 in human hepatocarcinoma cells.

    PubMed

    Cepeda, Edgar B; Dediulia, Tatjana; Fernando, Joan; Bertran, Esther; Egea, Gustavo; Navarro, Estanislao; Fabregat, Isabel

    2015-05-01

    Hepatocellular carcinoma (HCC) cells with a mesenchymal phenotype show an asymmetric subcellular distribution of the chemokine receptor CXCR4, which is required for cell migration and invasion. In this work we examine the mechanisms that regulate the intracellular trafficking of CXCR4 in HCC cells. Results indicate that HCC cells present CXCR4 at the cell surface, but most of this protein is in endomembranes colocalizing with markers of the Golgi apparatus and recycling endosomes. The presence of high protein levels of CXCR4 present at the cell surface correlates with a mesenchymal-like phenotype and a high autocrine activation of the Transforming Growth Factor-beta (TGF-β) pathway. CXCR4 traffics along the Golgi/exocyst/plasma membrane pathway and requires EXOC4 (Sec8) component of the exocyst complex. HCC cells use distinct mechanisms for the CXCR4 internalization such as dynamin-dependent endocytosis and macropinocytosis. Regardless of the endocytic mechanisms, colocalization of CXCR4 and Rab11 is observed, which could be involved not only in receptor recycling but also in its post-Golgi transport. In summary, this work highlights membrane trafficking pathways whose pharmacological targeting could subsequently result in the inactivation of one of the main guiding mechanisms used by metastatic cells to colonize secondary organs and tissues. PMID:25704914

  15. Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines

    PubMed Central

    Potenza, Nicoletta; Castiello, Filomena; Panella, Marta; Colonna, Giovanni; Ciliberto, Gennaro; Russo, Aniello; Costantini, Susan

    2016-01-01

    Hepatocellular carcinoma (HCC) is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK), whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3’UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3’UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium. PMID:27275761

  16. Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

    PubMed Central

    Rigalli, Juan Pablo; Ciriaci, Nadia; Arias, Agostina; Ceballos, María Paula; Villanueva, Silvina Stella Maris; Luquita, Marcelo Gabriel; Mottino, Aldo Domingo; Ghanem, Carolina Inés; Catania, Viviana Alicia; Ruiz, María Laura

    2015-01-01

    Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements. PMID:25781341

  17. Preclinical evaluation of azathioprine plus buthionine sulfoximine in the treatment of human hepatocarcinoma and colon carcinoma

    PubMed Central

    Hernández-Breijo, Borja; Monserrat, Jorge; Ramírez-Rubio, Sara; Cuevas, Eva P; Vara, Diana; Díaz-Laviada, Inés; Fernández-Moreno, M Dolores; Román, Irene D; Gisbert, Javier P; Guijarro, Luis G

    2011-01-01

    AIM: To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) by localized application into HepG2 tumor in vivo. METHODS: Different hepatoma and colon carcinoma cell lines (HepG2, HuH7, Chang liver, LoVo, RKO, SW-48, SW-480) were grown in minimal essencial medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution and maintained in a humidified 37 °C incubator with 5% CO2. These cells were pretreated with BSO for 24 h and then with AZA for different times. We examined the effects of this combination on some proteins and on cellular death. We also studied the efficacy and the safety of AZA (6 mg/kg per day) and BSO (90 mg/kg per day) in HepG2 tumor growth in vivo using athymic mice. We measured safety by serological markers such as aminotransferases and creatine kinase. RESULTS: The in vitro studies revealed a new mechanism of action for the AZA plus BSO combination in the cancer cells compared with other thiopurines (6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine and 6-methylthioguanine) in combination with BSO. The cytotoxic effect of AZA plus BSO in HepG2 cells resulted from necroptosis induction in a mitochondrial-dependent manner. From kinetic studies we suggest that glutathione (GSH) depletion stimulates c-Jun amino-terminal kinase and Bax translocation in HepG2 cells with subsequent deregulation of mitochondria (cytochrome c release, loss of membrane potential), and proteolysis activation leading to loss of membrane integrity, release of lactate dehydrogenase and DNA degradation. Some of this biochemical and cellular changes could be reversed by N-acetylcysteine (a GSH replenisher). In vivo studies showed that HepG2 tumor growth was inhibited when AZA was combined with BSO. CONCLUSION: Our studies suggest that a combination of AZA plus BSO could be useful for localized treatment of hepatocellular carcinoma as in the currently used transarterial chemoembolization method. PMID:22025878

  18. Combined cytotoxic effects of pesticide mixtures present in the Chinese diet on human hepatocarcinoma cell line.

    PubMed

    Ma, Mengmeng; Chen, Chen; Yang, Guiling; Li, Yun; Chen, Zhijun; Qian, Yongzhong

    2016-09-01

    Consumers might be simultaneously exposed to several pesticide residues contained in their food. Based on the results of previous studies, 20 pesticides were selected due to their high exposure levels to which the Chinese population is likely exposed through the diet. The purpose of this study was to measure the cytotoxicity of these pesticides in HepG2 cells in vitro, as an alternative approach to assess the toxicity of chemicals. Then, the pesticides and some of the mixtures with comparatively high cell-proliferating inhibitory activities were selected to test the cellular ROS level and apoptosis-related protein Caspase-3/7 content in HepG2 cells. The combined effects of these pesticide mixtures with the prediction was based on a combination index (CI)-isobologram equation and the pesticide combinations exhibited various types of interactions (synergism, antagonism, and additivity). Two individuals, one binary combinations, and three uniform design (UD) mixtures of the pesticides were found to have significant cytotoxic effects, along with significant time- and dose-dependent induction of caspase-3/7 activity in vitro, indicating that cytotoxicity caused by these pesticides might be attributed to the pro-oxidative and apoptosis induced potential. PMID:27300773

  19. Fibroblast Growth Factor Receptor 4 Polymorphism Is Associated with Liver Cirrhosis in Hepatocarcinoma

    PubMed Central

    Sheu, Ming-Jen; Hsieh, Ming-Ju; Chiang, Whei-Ling; Yang, Shun-Fa; Lee, Hsiang-Lin; Lee, Liang-Ming; Yeh, Chao-Bin

    2015-01-01

    Background Fibroblast growth factor receptor 4 (FGFR4) polymorphisms are positively correlated with tumor progression in numerous malignant tumors. However, the association between FGFR4 genetic variants and the risk of hepatocellular carcinoma (HCC) has not yet been determined. In this study, we investigated the potential associations of FGFR4 single nucleotide polymorphisms (SNPs) with HCC susceptibility and its clinicopathological characteristics. Methodology/Principal Findings Four SNPs in FGFR4 (rs1966265, rs351855, rs2011077, and rs7708357) were analyzed among 884 participants, including 595 controls and 289 patients with HCC. The samples were further analyzed to clarify the associations between these gene polymorphisms and the risk of HCC, and the impact of these SNPs on the susceptibility and clinicopathological characteristics of HCC. After adjusting for other covariants, HCC patients who carrying at least one A genotype (GA and AA) at rs351855 were observed to have a higher risk of liver cirrhosis compared with those carrying the wild-type genotype (GG) (OR: 2.113, 95% CI: 1.188–3.831). Moreover, the patients with at least one A genotype were particularly showed a high level of alpha-fetoprotein (AFP). Conclusions Our findings suggest that genetic polymorphism in FGFR4 rs351855 may be associated with the risk of HCC coupled with liver cirrhosis and may markedly increase the AFP level in Taiwanese patients with HCC. In addition, this is the first study that evaluated the risk factors associated with FGFR4 polymorphism variants in Taiwanese patients with HCC. PMID:25860955

  20. Clustering Nuclear Receptors in Liver Regeneration Identifies Candidate Modulators of Hepatocyte Proliferation and Hepatocarcinoma

    PubMed Central

    Graziano, Giusi; D'Orazio, Andria; Cariello, Marica; Massafra, Vittoria; Salvatore, Lorena; Martelli, Nicola; Murzilli, Stefania; Sasso, Giuseppe Lo; Mariani-Costantini, Renato; Moschetta, Antonio

    2014-01-01

    Background & Aims Liver regeneration (LR) is a valuable model for studying mechanisms modulating hepatocyte proliferation. Nuclear receptors (NRs) are key players in the control of cellular functions, being ideal modulators of hepatic proliferation and carcinogenesis. Methods & Results We used a previously validated RT-qPCR platform to profile modifications in the expression of all 49 members of the NR superfamily in mouse liver during LR. Twenty-nine NR transcripts were significantly modified in their expression during LR, including fatty acid (peroxisome proliferator-activated receptors, PPARs) and oxysterol (liver X receptors, Lxrs) sensors, circadian masters RevErbα and RevErbβ, glucocorticoid receptor (Gr) and constitutive androxane receptor (Car). In order to detect the NRs that better characterize proliferative status vs. proliferating liver, we used the novel Random Forest (RF) analysis to selected a trio of down-regulated NRs (thyroid receptor alpha, Trα; farsenoid X receptor beta, Fxrβ; Pparδ) as best discriminators of the proliferating status. To validate our approach, we further studied PPARδ role in modulating hepatic proliferation. We first confirmed the suppression of PPARδ both in LR and human hepatocellular carcinoma at protein level, and then demonstrated that PPARδ agonist GW501516 reduces the proliferative potential of hepatoma cells. Conclusions Our data suggest that NR transcriptome is modulated in proliferating liver and is a source of biomarkers and bona fide pharmacological targets for the management of liver disease affecting hepatocyte proliferation. PMID:25116592

  1. The NKD1/Rac1 feedback loop regulates the invasion and migration ability of hepatocarcinoma cells.

    PubMed

    Li, Jie; Zhang, Sheng; Hu, Qing; Zhang, Kang; Jin, Jianbin; Zheng, Xuqing; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatocellular carcinoma (HCC) is complicated by aggressive migration and invasion, which contribute to the increased mortality of HCC patients. The NKD1 protein is abnormally expressed in many neoplasms and plays an important role in tumor progression. However, the regulation and underlying molecular mechanisms of NKD1 in HCC cell invasion and migration remain poorly understood. In the present study, ectopic expression of NKD1 in HCC cells attenuated migration and invasion in vitro and in vivo by down-regulating Rac1 expression level and activity, which affected the HCC cell cytoskeleton and E-cadherin expression. Mechanistic studies showed that NKD1 interacted with Rac1 in the cytoplasm and promoted its degradation by the ubiquitin-proteasome pathway. Over-expression of Rac1 enhanced the transcription of the NKD1 gene and protein expression conversely owing to its negative regulation of EZH2. Analysis of clinical samples showed that abnormal expression of NKD1 and Rac1 was associated with the poor prognosis of HCC patients. In summary, our data indicate a new role for NKD1 as a regulator of HCC cell invasion and migration via a feedback loop involving Rac1. PMID:27231134

  2. The NKD1/Rac1 feedback loop regulates the invasion and migration ability of hepatocarcinoma cells

    PubMed Central

    Li, Jie; Zhang, Sheng; Hu, Qing; Zhang, Kang; Jin, Jianbin; Zheng, Xuqing; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatocellular carcinoma (HCC) is complicated by aggressive migration and invasion, which contribute to the increased mortality of HCC patients. The NKD1 protein is abnormally expressed in many neoplasms and plays an important role in tumor progression. However, the regulation and underlying molecular mechanisms of NKD1 in HCC cell invasion and migration remain poorly understood. In the present study, ectopic expression of NKD1 in HCC cells attenuated migration and invasion in vitro and in vivo by down-regulating Rac1 expression level and activity, which affected the HCC cell cytoskeleton and E-cadherin expression. Mechanistic studies showed that NKD1 interacted with Rac1 in the cytoplasm and promoted its degradation by the ubiquitin-proteasome pathway. Over-expression of Rac1 enhanced the transcription of the NKD1 gene and protein expression conversely owing to its negative regulation of EZH2. Analysis of clinical samples showed that abnormal expression of NKD1 and Rac1 was associated with the poor prognosis of HCC patients. In summary, our data indicate a new role for NKD1 as a regulator of HCC cell invasion and migration via a feedback loop involving Rac1. PMID:27231134

  3. Gum arabic-curcumin conjugate micelles with enhanced loading for curcumin delivery to hepatocarcinoma cells.

    PubMed

    Sarika, P R; James, Nirmala Rachel; Kumar, P R Anil; Raj, Deepa K; Kumary, T V

    2015-12-10

    Curcumin is conjugated to gum arabic, a highly water soluble polysaccharide to enhance the solubility and stability of curcumin. Conjugation of curcumin to gum arabic is confirmed by (1)H NMR, fluorescence and UV spectroscopy studies. The conjugate self assembles to spherical nano-micelles (270 ± 5 nm) spontaneously, when dispersed in aqueous medium. Spherical morphology of the self assembled conjugate is evidenced by field emission scanning electron microscopy and transmission electron microscopy. The self assembly of the amphiphilic conjugate into micelle in aqueous medium significantly enhances the solubility (900 fold of that of free curcumin) and stability of curcumin in physiological pH. The anticancer activity of the conjugate micelles is found to be higher in human hepatocellular carcinoma (HepG2) cells than in human breast carcinoma (MCF-7) cells. The conjugate exhibits enhanced accumulation and toxicity in HepG2 cells due to the targeting efficiency of the galactose groups present in gum arabic. PMID:26428113

  4. Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells

    SciTech Connect

    Mizumura, Ayano; Watanabe, Takayuki; Kobayashi, Yayoi; Hirano, Seishiro

    2010-01-15

    It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione-conjugated arsenicals can still react with some proteins in hepatic cells.

  5. Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines.

    PubMed

    Potenza, Nicoletta; Castiello, Filomena; Panella, Marta; Colonna, Giovanni; Ciliberto, Gennaro; Russo, Aniello; Costantini, Susan

    2016-01-01

    Hepatocellular carcinoma (HCC) is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK), whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3'UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3'UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium. PMID:27275761

  6. Lipid nanocarriers containing sorafenib inhibit colonies formation in human hepatocarcinoma cells.

    PubMed

    Bondì, Maria Luisa; Botto, Chiara; Amore, Erika; Emma, Maria Rita; Augello, Giuseppa; Craparo, Emanuela Fabiola; Cervello, Melchiorre

    2015-09-30

    Here, the potential of two nanostructured lipid carriers (NLC) for controlled release of sorafenib was evaluated. The obtained systems showed characteristics suitable as drug delivery systems for the treatment of hepatocellular carcinoma (HCC) through parenteral administration. The use of a mixture between a solid lipid (tripalmitin) with a liquid lipid (Captex 355 EP/NF or Miglyol 812) to prepare NLC systems could give a higher drug loading capacity and a longer term stability during storage than that obtained by using only solid lipids. The obtained nanoparticles showed a nanometer size and high negative zeta potential values. Scansion electron microscopy (SEM) of the sorafenib loaded NLC revealed a spherical shape with a diameter <300 nm. In vitro biological studies demonstrated that sorafenib loaded into NLC had enhanced anti-tumor activity compared to that of free drug. This finding raises hope in terms of future drug delivery strategy of sorafenib loaded NLC, that can be useful for therapeutic application in HCC. PMID:26211902

  7. Transarterial radioembolization vs chemoembolization for hepatocarcinoma patients: A systematic review and meta-analysis

    PubMed Central

    Facciorusso, Antonio; Serviddio, Gaetano; Muscatiello, Nicola

    2016-01-01

    AIM: To compare the efficacy and safety of yttrium-90 radioembolization (Y90RE) and transarterial chemoembolization (TACE) in hepatocellular carcinoma patients. METHODS: Bibliographic research was conducted on main scientific databases. When there was no statistically significant heterogeneity, pooled effects were calculated using a fixed-effects model by means of Mantel-Haenszel test, otherwise, a random-effects model was used with DerSimonian and Laird test. Summary estimates were expressed in terms of odds ratios (ORs) and 95%CI. The probability of publication bias was assessed using funnel plots and with Begg and Mazumdar’s test. Sensitivity analysis was finally conducted using the method of excluding extreme data. RESULTS: A total of 10 studies were analyzed, of which 2 randomized controlled trials. Survival rate (SR) assessed at 1 year showed an absolute similarity between the two treatment groups (OR = 1.01, 95%CI: 0.78-1.31, P = 0.93). As long as time elapsed since the treatment, ORs for survival rate tended to significantly increase, thus meaning better long-term outcomes in patients who underwent Y90RE (2-year SR: OR = 1.43, 1.08-1.89, P = 0.01; 3-year SR: OR = 1.48, 1.03-2.13, P = 0.04). Meta-analysis of plotted hazard ratios (HRs) determined a non-significant overall estimate in favor of Y90RE (HR = 0.91, 0.80-1.04, P = 0.16). Y90RE showed a statistically significant benefit as compared to TACE in terms of higher progression-free survival rate assessed at 1 year (OR = 1.67; 95%CI: 1.10-2.55; P = 0.02). Pooled analyses do not revealed a statistically significant increase in OR for tumor objective responses after Y90RE with respect to TACE (OR = 1.22, 95%CI: 0.69-2.16, P = 0.50). A non-significant trend in favor of Y90RE was observed according to adverse event rate (OR = 0.70, 0.38-1.30, P = 0.26). CONCLUSION: Our meta-analysis reveals that Y90RE and TACE show similar effects in terms of survival, response rate and safety profile, although tumor progression is delayed after radioembolization. PMID:27366304

  8. From minimal to maximal surgery in the treatment of hepatocarcinoma: A review.

    PubMed

    Perini, Marcos Vinicius; Starkey, Graham; Fink, Michael A; Bhandari, Ramesh; Muralidharan, Vijayaragavan; Jones, Robert; Christophi, Christopher

    2015-01-27

    Hepatocellular carcinoma represents one of the most challenging frontiers in liver surgery. Surgeons have to face a broad spectrum of aspects, from the underlying liver disease to the new surgical techniques. Safe liver resection can be performed in patients with portal hypertension and well-compensated liver function with a 5-year survival rate of 50%, offering good long-terms results in selected patients. With the advances in laparoscopic surgery, major liver resections can be performed with minimal harm, avoiding the wound and leak complications related to the laparotomies. Studies have shown that oncological margins are the same as in open surgery. In patients submitted to liver resection (either laparoscopic or open) who experience recurrence, re-resection or salvage liver transplantation has been showing to be an alternative approach in well selected cases. The decision making approach to the cirrhotic patient is becoming more complex and should involve hepatologists, liver surgeons, radiologists and oncologists. Better understanding of the different risk factors for recurrence and survival should be aimed in these multidisciplinary discussions. We here in discuss the hot topics related to surgical risk factors regarding the surgical treatment of hepatocellular carcinoma: anatomical resection, margin status, macrovascular tumor invasion, the place of laparoscopy, salvage liver transplantation and liver transplantation. PMID:25625000

  9. Molecular targeting agents associated with transarterial chemoembolization or radiofrequency ablation in hepatocarcinoma treatment

    PubMed Central

    Ranieri, Girolamo; Marech, Ilaria; Lorusso, Vito; Goffredo, Veronica; Paradiso, Angelo; Ribatti, Domenico; Gadaleta, Cosmo Damiano

    2014-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer in the world. According to Barcelona Clinic Liver Cancer modified criteria, patients with early stage disease are candidate to radiofrequency ablation (RFA), while patients with intermediate stage HCC are usually treated by transarterial chemoembolization (TACE). TACE and RFA induce a transient devascularisation effect followed by strong neo-angiogenic stimulus. In fact, after these procedures, it has been demonstrated an up-regulation of pro-angiogenic and growth factors such as vascular endothelial growth factor-A, which might contribute to accelerated progression in patients with incomplete response. Several studies have demonstrated that MAP-kinase and AKT pathways, in addition to neo-angiogenesis, have an important role in the development of HCC. In advanced HCC, anti-angiogenic therapy and tyrosine kinases inhibitors showed potential clinical benefit. Actually, a number of clinical studies are ongoing testing these agents in combination with TACE or RFA. In this paper, we have reviewed the most recent preclinical and clinical results of such trials. PMID:24574717

  10. The histone demethylase JMJD1A regulates adrenomedullin-mediated cell proliferation in hepatocellular carcinoma under hypoxia

    SciTech Connect

    Park, Seong-Joon; Kim, Joong-Gook; Son, Tae Gen; Yi, Joo Mi; Kim, Nam Deuk; Yang, Kwangmo; Heo, Kyu

    2013-05-17

    Highlights: •Hypoxia stimulates HepG2 and Hep3B cell proliferation. •The JMJD1A and ADM expressions are enhanced by hypoxia in HCCs. •Increased JMJD1A expression reduces a H3K9 di-methylation in the ADM promoter region. •Knock-down of JMJD1A abrogates the hypoxia-induced HepG2 and Hep3B cell growth. -- Abstract: We studied the roles of JMJD1A and its target gene ADM in the growth of hepatocellular carcinomas (HCCs) and breast cancer cells under hypoxic conditions. Hypoxia stimulated HepG2 and Hep3B cell proliferation but had no effect on MDA-MB-231 cell proliferation. Interestingly, the JMJD1A and ADM expressions were enhanced by hypoxia only in HepG2 and Hep3B cells. Our ChIP results showed that hypoxia-induced HepG2 and Hep3B cell proliferation is mediated by JMJD1A upregulation and subsequent decrease in methylation in the ADM promoter region. Furthermore, JMJD1A gene silencing abrogated the hypoxia-induced ADM expression and inhibited HepG2 and Hep3B cell growth. These data suggest that JMJD1A might function as a proliferation regulator in some cancer cell types.

  11. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  12. Primary seronegative but molecularly evident hepadnaviral infection engages liver and induces hepatocarcinoma in the woodchuck model of hepatitis B.

    PubMed

    Mulrooney-Cousins, Patricia M; Chauhan, Ranjit; Churchill, Norma D; Michalak, Tomasz I

    2014-08-01

    Hepadnavirus at very low doses establishes in woodchucks asymptomatic, serologically undetectable but molecularly evident persistent infection. This primary occult infection (POI) preferentially engages the immune system and initiates virus-specific T cell response in the absence of antiviral antibody induction. The current study aimed to determine whether POI with time may culminate in serologically identifiable infection and hepatitis, and what are, if any, its pathological consequences. Juvenile woodchucks were intravenously injected with inocula containing 10 or 100 virions of woodchuck hepatitis virus (WHV) to induce POI and followed for life or up to 5.5 years thereafter. All 10 animals established molecularly detectable infection with virus DNA in serum (<100-200 copies/mL) and in circulating lymphoid cells, but serum WHV surface antigen and antibodies to WHV core antigen remained undetectable for life. By approximately 2.5-3.5 years post-infection, circulating virus transiently increased to 103 copies/mL and virus replication became detectable in the livers, but serological markers of infection and biochemical or histological evidence of hepatitis remained undetectable. Nonetheless, typical hepatocellular carcinoma (HCC) developed in 2/10 animals. WHV DNA integration into hepatic and lymphatic system genomes was identified in 9/10 animals. Virus recovered from the liver virus-negative or virus-positive phases of POI displayed the wild-type sequence and transmitted infection to healthy woodchucks causing hepatitis and HCC. In summary, for the first time, our data demonstrate that an asymptomatic hepadnaviral persistence initiated by very small amounts of otherwise pathogenic virus, advancing in the absence of traditional serological markers of infection and hepatitis, coincides with virus DNA integration into the host's hepatic and immune system genomes, retains liver pro-oncogenic potency and is capable of transmitting liver pathogenic infection. This emphasizes the role for primary occult hepatitis B virus infection in the development of seemingly cyptogenic HCC in seronegative but virus DNA reactive patients. PMID:25165821

  13. Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line

    PubMed Central

    Teimourpour, Roghayeh; Zare, Hosna; Rajabnia, Ramazan; Yahyapour, Yousef; Meshkat, Zahra

    2016-01-01

    Background and Objectives: HBHA and Mtb32C have been isolated from culture supernatants of Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis) and their immunogenicity previously studies have been confirmed. In this study, capability of constructed vector containing two mycobacterial immunodaminant antigens (Mtb32C-HBHA), in producing new chimeric protein under the in-vitro condition was examined. Materials and Methods: In present study Huh7.5 cells was transfected with Mtb32C-HBHA −pCDNA3.1+ recombinant vector using the calcium phosphate method and expression of chimeric protein was assessed by RT-PCR and Western blot methods. Results: Results of RT-PCR and Western blot showed expression of 35.5 KD recombinant protein (Mtb32C-HBHA) in this cell line. Conclusion: The constructed vector can produce two highly immunogenic antigens that fusion of them to gather makes chimeric antigen with new traits. Other attempts are needed to evaluate specific properties of this new antigen such as molecular conformation modeling and immunologic characteristics in future studies. PMID:27307979

  14. Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

    SciTech Connect

    Yang, H.; Gan, L.; Yang, X. E-mail: yangxl@mail.hust.edu.cn; Lu, R.; Xian, Y.; Lu, X. E-mail: yangxl@mail.hust.edu.cn

    2015-12-15

    Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-kB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

  15. Isolation and purification of schisandrol A from the stems of Schisandra chinensis and cytotoxicity against human hepatocarcinoma cell lines

    PubMed Central

    Zhu, Lijie; Li, Bin; Liu, Xiuying; Huang, Guohui; Meng, Xianjun

    2015-01-01

    Background: Schisandrol A, a lignan with anticancer effects, is one of the representative components that identifies Schisandra chinensis. Objective: A method for purifying schisandrol A from the stems of S. chinensis was established using an octadecylsilyl (ODS) column combined with preparative high-performance liquid chromatography (HPLC). Materials and Methods: Crude extracts obtained from the stems of S. chinensis using 70% ethanol were separated on an AB-8 macroporous resin column and then eluted with a graded ethanol series. After 70% methanol was used in an ODS column separation, preparative HPLC was used for subsequent purification. The structure was identified by electrospray ionization-mass spectrometry, 1H-nuclear magnetic resonance, and 13C-nuclear magnetic resonance. HepG2 and Bel-7402 hepatocellular carcinoma cell lines were used for toxicological evaluation. Results: 21.4 mg of schisandrol A with a purity of 95.2% were collected. The cytotoxicity of the ODS-purified sample and schisandrol A were significantly stronger than that of a resin-purified sample. Conclusion: Schisandrol A was successfully extracted from the stems of S. chinensis and separated with an ODS column combined with preparative HPLC. The samples obtained during the purification process showed different levels of cytotoxicity on the HepG2 and Bel-7402 hepatocellular carcinoma cell lines. PMID:25709222

  16. Generation of reactive oxygen species by a novel berberine–bile acid analog mediates apoptosis in hepatocarcinoma SMMC-7721 cells

    SciTech Connect

    Li, Qingyong; Zhang, Li; Zu, Yuangang; Liu, Tianyu; Zhang, Baoyou; He, Wuna

    2013-04-19

    Graphical abstract: - Highlights: • Anticancer effects of B4, a novel berberine–bile acid analog, were tested. • B4 inhibited cell proliferation in hepatocellular carcinoma cells. • It also stimulated mitochondrial ROS production and membrane depolarization. • Effects of B4 were inhibited by a non-specific ROS scavenger. • Regulation of ROS generation may be a strategy for treating hepatic carcinoma. - Abstract: 2,3-Methenedioxy-9-O-(3′α,7′α-dihydroxy-5′β-cholan-24′-propy-lester) berberine (B4) is a novel berberine–bile acid analog synthesized in our laboratory. Previously, we showed that B4 exerted greater cytotoxicity than berberine in several human cancer cell lines. Therefore, we further evaluated the mechanism governing its anticancer actions in hepatocellular carcinoma SMMC-7721 cells. B4 inhibited the proliferation of SMMC-7721 cells, and stimulated reactive oxygen species (ROS) production and mitochondrial membrane depolarization; anti-oxidant capacity was reduced. B4 also induced the release of cytochrome c from the mitochondria to the cytosol and an increase in poly ADP-ribose polymerase (PARP) cleavage products, reflective of caspase-3 activation. Moreover, B4 induced the nuclear translocation of apoptosis-inducing factor (AIF) and a rise in DNA fragmentation. Pretreatment with the anti-oxidant N-acetylcysteine (NAC) inhibited B4-mediated effects, including cytotoxicity, ROS production, mitochondrial membrane depolarization increase in intracellular Ca{sup 2+}, cytochrome c release, PARP cleavage, and AIF translocation. Our data suggest that B4 induces ROS-triggered caspase-dependent and caspase-independent apoptosis pathways in SMMC-7721 cells and that ROS production may be a specific potential strategy for treating hepatic carcinoma.

  17. RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation.

    PubMed

    Bouhlal, Hicham; Ouled-Haddou, Hakim; Debuysscher, Véronique; Singh, Amrathlal Rabbind; Ossart, Christèle; Reignier, Aline; Hocini, Hakim; Fouquet, Gregory; Al Baghami, Mohammed; Eugenio, Mélanie Simoes; Nguyen-Khac, Eric; Regimbeau, Jean-Marc; Marcq, Ingrid

    2016-03-01

    Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression. PMID:26799423

  18. Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

    PubMed

    Liu, W; Wang, J; Wang, L; Qian, C; Qian, Y; Xuan, H; Zhuo, W; Li, X; Yu, J; Si, J

    2016-01-01

    Ras-Association Domain Family 10 (RASSF10) is the last identified member of the RASSF family. The functional characteristics of this new gene in human cancers remain largely unclear. Here, we examined RASSF10 for the biological functions and related molecular mechanisms in hepatocellular carcinoma (HCC). We found that RASSF10 is expressed in normal human liver tissue, but is silenced or down-regulated in 62.5% (5/8) of HCC cell lines. The mean expression level of RASSF10 was significantly lower in primary HCCs compared with their adjacent normal tissues (P<0.005, n=52). The promoter methylation contributes to the inactivation of RASSF10 as demonstrated by bisulfite genomic sequencing and demethylation treatment analyses. Transgenic expression of RASSF10 in silenced HCC cell lines suppressed cell viability, colony formation and inhibited tumor growth in nude mice (QGY7703, P<0.01; HepG2, P<0.05). Furthermore, RASSF10 was shown to induce the cell accumulation in G1 phase with the increase of p27, as well as the decrease of cyclinD1 and CDK2/CDK4. Over-expression of RASSF10 also inhibited HCC cells migration (P<0.01) or invasion (P<0.05). Adhesion genes array revealed that Matrix Metalloproteinase 2 (MMP2) was a downstream effector of RASSF10. RASSF10 acting as a tumor suppressor to inhibit HCC invasion partially mediated by Focal Adhesion Kinase or p38 MAPK to decrease the accumulation of MMP2. Our study suggests that RASSF10 acts as a tumor suppressor for HCC. PMID:27348267

  19. Complete regression of a guinea pig hepatocarcinoma by immunotherapy with "tumor-immune" RNA or antibody to fibrin fragment E.

    PubMed

    Schlager, S I; Dray, S

    1976-01-01

    Two novel immunotherapeutic regimens were developed for a uniformly lethal, intradermally growing transplantable ascites variant (line 10) of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. In an apparently tumor-specific immunotherapy model, 32 guinea pigs were cured by the injection into the tumor area, five or seven days after tumor challenge, of syngeneic or xenogeneic RNA extracts obtained from lymphoid tissues of line 10-immune strain 2 guinea pigs or rhesus monkeys, as part of a total regimen which included syngeneic nonsensitive peritoneal exudate cells injected prior to, and tumor-specific antigen injected after, the RNA. In another immunotherapy model, not tumor-specific, 18 strain 2 guinea pigs were cured by the injection into the tumor area, 6 and 16 days after tumor challenge, of antibody specific for fibrin fragment E (FFE), an essential component in the formation of a fibrin matrix considered to be important in tumor development. When therapy was delayed to 12 days in the RNA test system, or to 16 days in the anti-FFE test system, complete abrogation of the tumors did not occur. The long-term survival of the 50 successfully treated animals and their immunity to further tumor challenge indicated that both immunotherapeutic procedures had systemic effects. To test this further, line 10 cells were injected intradermally simultaneously at two sites and only one site was treated. When the one tumor location was treated with anti-FFE, complete regression of the treated tumor and a 30% retardation in the development of the untreated tumor were observed. When this tumor location was treated with the RNA regimen, complete regression of the tumors occurred at both the treated and the untreated sites. Optimal conditions for both immunotherapeutic models and their combination have yet to be establshed. Nonetheless, both immunotherapeutic regimens were more effective than any other immunotherapy thus far reported for this tumor, including the use of BCG or its derivatives. PMID:181352

  20. Corosolic acid analogue, a natural triterpenoid saponin, induces apoptosis on human hepatocarcinoma cells through mitochondrial pathway in vitro.

    PubMed

    Qu, Liping; Zhang, Huiqing; Yang, Yanlong; Yang, Geliang; Xin, Hailiang; Ling, Changquan

    2016-08-01

    Context 2a,-3a,-24-Trihydroxyurs-12-en-28-oic acid (TEO, a corosolic acid analogue) is a triterpenoid saponin isolated from Actinidia valvata Dunn (Actinidiaceae), a well-known traditional Chinese medicine. Objective This study investigated the anti-proliferation and inducing apoptosis effects of TEO in three human hepatocellular carcinoma (HCC) cell lines. Materials and methods Cytotoxic activity of TEO was determined by the MTT assay at various concentrations from 2.5 to 40 μg/mL in BEL-7402, BEL-7404 and SMMC-7721 cell lines. Cell morphology was assessed by acridine orange/ethidium bromide and 4'-6-diamidino-2-phenylindole dihydrochloride staining and fluorescence microscopy. Cell-cycle distribution and DNA damage were determined by flow cytometry and comet assay. Mitochondrial dysfunction was assessed by JC-1 staining and transmission electron microscopy. Apoptosis changes were explored by Western blot, TNF-α and caspase-3, -8, -9 assays. Results TEO exhibited inhibition effects on BEL-7402, BEL-7404 and SMMC-7721 cells treated for 24 h, the IC50 values were 34.6, 30.8 and 30.5 μg/mL, respectively. TEO (40 μg/mL)-treated three cell lines increased by more than 21% in the G1 phase and presented the morphological change and DNA damage. TEO also declined the mitochondrial membrane potential and altered mitochondrial ultra-structure. Furthermore, caspase-3, caspase-8, caspase-9 and TNF-α were also activated. Mechanism investigation showed that TEO could decrease anti-apoptotic Bcl-2 protein expression, increase proapoptotic Bax and Bid proteins expressions and increase Bax/Bcl-2 ratio. Conclusion Our results demonstrate for the first time that TEO inhibited growth of HCC cell lines and induced G1 phase arrest. Moreover, proapoptotic effects of TEO were mediated through the activation of TNF-α, caspases and mitochondrial pathway. PMID:26810384

  1. Transcriptional repression of MMP-1 by p21SNFT and reduced in vitro invasiveness of hepatocarcinoma cells.

    PubMed

    Bower, Kristen E; Fritz, Jamie M; McGuire, Kathleen L

    2004-11-18

    p21SNFT (21 kDa small nuclear factor isolated from T cells) is a human basic leucine zipper transcription factor that can repress AP-1-mediated transcription. We show here that overexpression of p21SNFT in HepG2 cells leads to repression of matrix metalloproteinase-1 by 70-80%. p21SNFT interacted with Jun at the matrix metalloproteinase-1 promoter -88 Ets/AP-1 enhancer element, where Jun is known to activate transcription via interaction with Fos and Ets proteins. When p21SNFT/Jun dimers bound the element in the presence of Ets, DNA was protected differently than when Fos was paired with Jun. The data suggest a difference in overall conformation between p21SNFT-containing and Fos-containing complexes that may be involved in the repression of matrix metalloproteinase-1 by p21SNFT. Overexpression of p21SNFT led to a reduction in invasiveness of HepG2 cells through type I collagen and reconstituted basement membrane, an effect similar to that obtained via direct immunodepletion of matrix metalloproteinase-1. The results indicate that the mechanism of repression of matrix metalloproteinase-1 by p21SNFT may be exploited in inhibiting pathological matrix remodeling during cancer progression in vivo. PMID:15467742

  2. Galectin-1 Controls the Proliferation and Migration of Liver Sinusoidal Endothelial Cells and Their Interaction With Hepatocarcinoma Cells.

    PubMed

    Manzi, Malena; Bacigalupo, María L; Carabias, Pablo; Elola, María T; Wolfenstein-Todel, Carlota; Rabinovich, Gabriel A; Espelt, María V; Troncoso, María F

    2016-07-01

    Galectin-1 (Gal1), a β-galactoside-binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial-mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness, and metastasis. During the early stages of HCC, transforming growth factor β1 (TGF-β1 ) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF-β1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF-β1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH-7 human HCC cells, and in SK-HEP-1 human liver sinusoidal endothelial cells (SECs). We used loss-of-function and gain-of-function experiments to down- or up-regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK-HEP-1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch-wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan-dependent heterotypic adhesion of HepG2 cells to SK-HEP-1 SECs. Furthermore, TGF-β1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK-HEP-1 SECs through a Gal1-dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF-β1 -induced growth inhibition. Our results suggest that Gal1 and TGF-β1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis, and angiogenesis. J. Cell. Physiol. 231: 1522-1533, 2016. © 2015 Wiley Periodicals, Inc. PMID:26551914

  3. Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

    NASA Astrophysics Data System (ADS)

    Yang, H.; Lu, R.; Xian, Y.; Gan, L.; Lu, X.; Yang, X.

    2015-12-01

    Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-кB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

  4. Radiation Exposure Promotes Hepatocarcinoma Cell Invasion through Epithelial Mesenchymal Transition Mediated by H2S/CSE Pathway.

    PubMed

    Pan, Yan; Zhou, Cuiping; Yuan, Dexiao; Zhang, Jianghong; Shao, Chunlin

    2016-01-01

    There is growing evidence to suggest that radiotherapy can paradoxically promote tumor invasion and metastatic processes, however, the underlying molecular mechanisms remain obscure. In this study, we found that exposure to X rays promoted cell invasion by triggering the epithelial mesenchymal transition (EMT) in two hepatocellular carcinoma (HCC) cell lines, HepG2 and PLC/PRF/5. This was made evident by a reduced expression of E-cadherin and enhanced expressions of N-cadherin, Vimentin and Snail. Moreover, exposure to radiation stimulated the signaling of hydrogen sulfide (H2S), a newly found gas transmitter, by upregulating the expressions of H2S-producing proteins of cysthionine-γ-lyase (CSE), cystathionine-β-synthase (CBS). Inhibition of CSE by siRNA or inhibitor not only increased the radiosensitivity but also strongly suppressed radiation-enhanced invasive properties of HCC cells. Interestingly, we found that H2S/CSE inhibition attenuated radiation-enhanced EMT, and the above effect was an end result of blockage of the radiation-activated pathway of p38 mitogen-activated protein kinase (p38MAPK). Collectively, our findings indicate that radiation could promote HCC cell invasion through EMT mediated by endogenous H2S/CSE signaling via the p38MAPK pathway. PMID:26727544

  5. Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

    PubMed Central

    Valdameri, Glaucio; Rocha, Maria Eliane Merlin; Martinez, Glaucia Regina; Noleto, Guilhermina Rodrigues; Acco, Alexandra; Alves de Souza, Carlos Eduardo; Echevarria, Aurea; Moretto dos Reis, Camilla; Di Pietro, Attilio; Suter Correia Cadena, Sílvia Maria

    2015-01-01

    In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma. PMID:26083249

  6. Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

    PubMed

    Gozzi, Gustavo Jabor; Pires, Amanda do Rocio Andrade; Valdameri, Glaucio; Rocha, Maria Eliane Merlin; Martinez, Glaucia Regina; Noleto, Guilhermina Rodrigues; Acco, Alexandra; Alves de Souza, Carlos Eduardo; Echevarria, Aurea; Moretto Dos Reis, Camilla; Di Pietro, Attilio; Suter Correia Cadena, Sílvia Maria

    2015-01-01

    In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma. PMID:26083249

  7. A nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis

    PubMed Central

    Chao, Ming; Wu, Hao; Jin, Kai; Li, Bin; Wu, Jianjun; Zhang, Guangqiang; Yang, Gong; Hu, Xun

    2016-01-01

    Study design: Previous works suggested that neutralizing intratumoral lactic acidosis combined with glucose deprivation may deliver an effective approach to control tumor. We did a pilot clinical investigation, including a nonrandomized (57 patients with large HCC) and a randomized controlled (20 patients with large HCC) studies. Methods: The patients were treated with transarterial chemoembolization (TACE) with or without bicarbonate local infusion into tumor. Results: In the nonrandomized controlled study, geometric mean of viable tumor residues (VTR) in TACE with bicarbonate was 6.4-fold lower than that in TACE without bicarbonate (7.1% [95% CI: 4.6%–10.9%] vs 45.6% [28.9%–72.0%]; p<0.0001). This difference was recapitulated by a subsequent randomized controlled study. TACE combined with bicarbonate yielded a 100% objective response rate (ORR), whereas the ORR treated with TACE alone was 44.4% (nonrandomized) and 63.6% (randomized). The survival data suggested that bicarbonate may bring survival benefit. Conclusion: Bicarbonate markedly enhances the anticancer activity of TACE. Clinical trail registration: ChiCTR-IOR-14005319. DOI: http://dx.doi.org/10.7554/eLife.15691.001 PMID:27481188

  8. RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation

    PubMed Central

    Bouhlal, Hicham; Singh, Amrathlal Rabbind; Ossart, Christèle; Reignier, Aline; Hocini, Hakim; Fouquet, Gregory; Baghami, Mohammed Al; Eugenio, Mélanie Simoes; Nguyen-Khac, Eric; Regimbeau, Jean-Marc; Marcq, Ingrid

    2016-01-01

    Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression. PMID:26799423

  9. Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma

    PubMed Central

    Liu, W; Wang, J; Wang, L; Qian, C; Qian, Y; Xuan, H; Zhuo, W; Li, X; Yu, J; Si, J

    2016-01-01

    Ras-Association Domain Family 10 (RASSF10) is the last identified member of the RASSF family. The functional characteristics of this new gene in human cancers remain largely unclear. Here, we examined RASSF10 for the biological functions and related molecular mechanisms in hepatocellular carcinoma (HCC). We found that RASSF10 is expressed in normal human liver tissue, but is silenced or down-regulated in 62.5% (5/8) of HCC cell lines. The mean expression level of RASSF10 was significantly lower in primary HCCs compared with their adjacent normal tissues (P<0.005, n=52). The promoter methylation contributes to the inactivation of RASSF10 as demonstrated by bisulfite genomic sequencing and demethylation treatment analyses. Transgenic expression of RASSF10 in silenced HCC cell lines suppressed cell viability, colony formation and inhibited tumor growth in nude mice (QGY7703, P<0.01; HepG2, P<0.05). Furthermore, RASSF10 was shown to induce the cell accumulation in G1 phase with the increase of p27, as well as the decrease of cyclinD1 and CDK2/CDK4. Over-expression of RASSF10 also inhibited HCC cells migration (P<0.01) or invasion (P<0.05). Adhesion genes array revealed that Matrix Metalloproteinase 2 (MMP2) was a downstream effector of RASSF10. RASSF10 acting as a tumor suppressor to inhibit HCC invasion partially mediated by Focal Adhesion Kinase or p38 MAPK to decrease the accumulation of MMP2. Our study suggests that RASSF10 acts as a tumor suppressor for HCC. PMID:27348267

  10. The antiproliferative effect of Origanum majorana on human hepatocarcinoma cell line: suppression of NF-κB.

    PubMed

    Fathy, S A; Emam, M A; Abo Agwa, S H; Abu Zahra, F A; Youssef, F S; Sami, R M

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancer types with a high prevalence and itis the leading cause of cancer deaths worldwide. This study aimed to investigate the antiproliferative effect of aqueous and ethanol extracts of Origanum majorana leaf on human hepatocellular carcinoma (HepG2) cell line through incubation of various concentrations of origanum majorana extracts with HepG2 and at different time intervals. The effects of aqueous and ethanol extracts of O. majorana L. on HepG2 cell viability, nuclear factor kappa B (NF-kB) gene expression were examined. The results of the cell viability assays showed that aqueous and ethanol extracts exhibited a highly significant inhibitory effect on HepG2 cell proliferation which was evidenced by a reduction in viable cell count. The results were confirmed by microscopical examination of cell morphology. Furthermore, the O. majorana L. extracts suppressed the activity of NF-kB gene expression of HepG2 cells compared to the control.The conclusions from this study suggest that marjoram extracts exhibit anti-proliferative effect against HCC through suppressing the activity of NF-kB gene expression and high antioxidant activity. PMID:27609479

  11. Aptamer targeting of the elongation factor 1A impairs hepatocarcinoma cells viability and potentiates bortezomib and idarubicin effects.

    PubMed

    Scaggiante, Bruna; Farra, Rosella; Dapas, Barbara; Baj, Gabriele; Pozzato, Gabriele; Grassi, Mario; Zanconati, Fabrizio; Grassi, Gabriele

    2016-06-15

    The high morbidity and mortality of hepatocellular carcinoma (HCC) is mostly due to the limited efficacy of the available therapeutic approaches. Here we explore the anti-HCC potential of an aptamer targeting the elongation factor 1A (eEF1A), a protein implicated in the promotion of HCC. As delivery methods, we have compared the effectiveness of cationic liposome and cholesterol-mediated approaches. A75 nucleotide long aptamer containing GT repetition (GT75) was tested in three HCC cell lines, HepG2, HuH7 and JHH6. When delivered by liposomes, GT75 was able to effectively reducing HCC cells viability in a dose and time dependent fashion. Particular sensitive were JHH6 where increased apoptosis with no effects on cell cycle were observed. GT75 effect was likely due to the interference with eEF1A activity as neither the mRNA nor the protein levels were significantly affected. Notably, cholesterol-mediated delivery of GT75 abrogated its efficacy due to cellular mis-localization as proven by fluorescence and confocal microscopic analysis. Finally, liposome-mediated delivery of GT75 improved the therapeutic index of the anticancer drugs bortezomib and idarubicin. In conclusion, liposome but not cholesterol-mediated delivery of GT75 resulted in an effective delivery of GT75, causing the impairment of the vitality of a panel of HCC derived cells. PMID:27094354

  12. Lenguaje y communicacion humanos (Human Language and Communication)

    ERIC Educational Resources Information Center

    Thomas, Lewis

    1976-01-01

    This essay affirms that people are not, like insects, genetically compelled to live and work collectively. Above all, language separates us from the rest of the animal world, allowing us to depart from reflex activity to consider the ambiguous and unknown and formulate new ideas. (Text is in Spanish.) (CHK)

  13. HBX Protein-Induced Downregulation of microRNA-18a is Responsible for Upregulation of Connective Tissue Growth Factor in HBV Infection-Associated Hepatocarcinoma

    PubMed Central

    Liu, Xiaomin; Zhang, Yingjian; Wang, Ping; Wang, Hongyun; Su, Huanhuan; Zhou, Xin; Zhang, Lamei

    2016-01-01

    Background This study was designed to improve our understanding of the role of miR-18a and its target (connective tissue growth factor (CTGF), which are mediators in HBX-induced hepatocellular carcinoma (HCC). Material/Methods We first investigated the expression of several candidate microRNAs (miRNAs) reported to have been aberrantly expressed between HepG2 and HepG2.2.15, which is characterized by stable HBV infection, while the CTGF is identified as a target of miR-18a. Furthermore, the expression of CTGF evaluated in HepG2 was transfected with HBX, while the HepG2.2.15 was transfected with miR-18a and CTGF siRNA. We examined the cell cycle at the same time. Results We found that the expression of miR-18a was abnormally reduced in the HBV-positive HCC tissue samples compared with HBV-negative HCC samples. Through the use of a luciferase reporter system, we also identified CTGF 3′UTR (1046–1052 bp) as the exact binding site for miR-18a. We also observed a clear increase in CTGF mRNA and protein expression levels in HBV-positive HCC human tissue samples in comparison with the HBV-negative controls, indicating a possible negatively associated relationship between miR-18a and CTGF. Furthermore, we investigated the effect of HBX overexpression on miR-18a and CTGF, as well as the viability and cell cycle status of HepG2 cells. In addition, we found that HBX introduction downregulated miR-18a, upregulated CTGF, elevated the viability, and promoted cell cycle progression. We transfected HepG2.2.15 with miR-18a mimics and CTGF siRNA, finding that upregulated miR-18a and downregulated CTGF suppress the viability and cause cell cycle arrest. Conclusions Our study shows the role of the CTGF gene as a target of miR-18a, and identifies the function of HBV/HBX/miR-18a/CTGF as a key signaling pathway mediating HBV infection-induced HCC. PMID:27421245

  14. MiR-101 targets DUSP1 to regulate the TGF-β secretion in sorafenib inhibits macrophage-induced growth of hepatocarcinoma

    PubMed Central

    Wei, Xufu; Tang, Chengyong; Lu, Xiuxian; Liu, Rui; Zhou, Mi; He, Diao; Zheng, Daofeng; Sun, Chao; Wu, Zhongjun

    2015-01-01

    Hepatocellular carcinoma (HCC)-associated macrophages accelerate tumor progression via growth factor release. Therefore, tumor-associated macrophages (TAMs)-initiated signaling cascades are potential therapeutic targets. To better understand anticancer effects of systemic HCC therapy, we studied sorafenib's effect on macrophage function, focusing on macrophage-related growth factor secretion. We found that dual specificity phosphatase 1 (DUSP1) is a direct target of miR-101. Transfection of miR-101 reduced DUSP1 induction in M2 macrophages and prolonged ERK1/2, p38 and JNK activation, whereas inhibition of miR-101 enhanced DUSP1 expression and decreased ERK1/2, p38 and JNK activation. miR-101 expression was decreased by sorafenib, and inhibition of PI3K/AKT blocked induction of miR-101 by LPS in M2 cells. M2 cells with greater TGF-β and CD206 mRNA expression compared to M1 cells had increased hepatoma growth, metastases and EMT. Sorafenib inhibited miR-101 expression and enhanced DUSP1 expression and lowered TGF-β and CD206 release in M2 cells, slowing macrophage-driven HCC. Our studies demonstrate miR-101 regulates macrophage innate immune responses to LPS via targeting DUSP1. Sorafenib alters macrophage polarization, reduces TGF-β driven cancer growth, metastases and EMT in vitro, and partially inhibits macrophage activation in vivo. Thus, macrophage modulation might explain the anticancer effects of sorafenib. PMID:26158762

  15. Effect of hypoxia on UDP-glucuronosyl transferase mRNA expression in human hepatocarcinoma functional liver celL4 cell line.

    PubMed

    Kato, R; Matsura, A; Kamiya, R; Oishi, C; Kagawa, Y; Tanaka, F; Matsumoto, N; Ijiri, Y; Hayashi, T

    2016-03-01

    Although hypoxic conditions have been reported to affect the expression levels of various enzymes like cytochrome P450, the effect of hypoxia for UDP-glucuronosyl transferase (UGT) expression has been unclear. We evaluated the mRNA expression of UGTs (UGT1A1·1A6·1A9·2B7) in a functional liver cell-4 (FLC-4) cell line by three-dimensional culture under hypoxic conditions (37 °C, 1% O₂, 5% CO₂) fo 7 days. The mRNA expression of UGT1A1·1A6·1A9·2B7 decreased significantly after 3 days and that of UGT1A1·1A6·1A9 decreased significantly after 7 days. Hypoxic conditions affect the expression levels of UGT enzymes, thus the adjustment of dosage and interval should be considered in drug therapy that metabolized by UGT. PMID:27183710

  16. Human hepatocarcinoma functional liver cell-4 cell line exhibits high expression of drug-metabolizing enzymes in three-dimensional culture.

    PubMed

    Kato, Ryuji; Shigemoto, Kota; Akiyama, Hiromasa; Ieda, Asaka; Ijiri, Yoshio; Hayashi, Tetsuya

    2014-01-01

    The expression levels of CYP and uridine diphosphate-glucuronosyl transferase (UGT) are lower in hepatocellular carcinoma cell lines than in human primary hepatocytes. However, a functional liver cell (FLC)-4 cell line that has a greater capacity to secrete liver-specific proteins than other hepatocellular carcinoma cells has recently been established. A three-dimensional culture using Engelbreth-Holm-Swan (EHS) gel induces the secretion of liver-specific proteins via the induction of hepatocyte nuclear factor-4α (HNF-4α). The aim of this study was to evaluate the mRNA expression of the enzymes CYP and UGT in FLC-4 and HepG2 cells in monolayer and three-dimensional cultures using EHS gel. The mRNA levels of HNF-4α, albumin, pregnane X receptor (PXR), constitutive androstane receptor (CAR), CYPs (1A2, 2E1, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) were determined using real-time reverse transcription (RT) PCR. In a monolayer culture, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, and 1A9) were higher in FLC-4 cells than in HepG2 cells. In FLC-4 cells, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C8, 2C19, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) significantly increased in three-dimensional culture. FLC-4 cells cultured in EHS gel showed significantly increased expression levels of CYPs and UGTs. The results of this study suggest that human hepatocellular carcinoma FLC-4 cells cultured in EHS gel show potential for use in studying in vitro drug metabolism. PMID:25366484

  17. A quantitative HPLC-UV method for determination of serum sorafenib and sorafenib N-oxide and its application in hepatocarcinoma patients.

    PubMed

    Shimada, Miki; Okawa, Hoshimi; Maejima, Takahiro; Yanagi, Toshiki; Hisamichi, Kanehiko; Matsuura, Masaki; Akasaka, Kazutoshi; Tsuchiya, Masami; Kondo, Yasuteru; Shimosegawa, Tooru; Mori, Masaru; Maekawa, Masamitsu; Suzuki, Hiroyuki; Mano, Nariyasu

    2014-01-01

    Sorafenib, an oral multi-kinase inhibitor, has been approved for treatment of advanced renal-cell and hepatocellular carcinoma (HCC). However, 20% of HCC patients taking sorafenib are forced to withdraw due to adverse effects within one month after administration. Orally administered sorafenib is oxidatively metabolized, predominantly by cytochrome P450 3A4 (CYP3A4), in small-intestinal mucosa or liver. We aimed to characterize the CYP3A4-mediated metabolism of sorafenib in HCC patients and explore the contribution of the major metabolite sorafenib N-oxide to adverse effects and therapeutic efficacy. We have therefore developed a method for quantitative determination of sorafenib and its N-oxide in the present study. To optimize the preanalytical procedure, we initially ascertained the solubility of the analytes. Because they are lipophilic, solvents containing more than 40% acetonitrile were required for efficient recovery. The pretreatment procedure that we ultimately developed consists of acetonitrile precipitation, followed by extraction using octadecyl silyl-silica gel to eliminate water-soluble and hydrophilic components of serum. Application of this procedure before HPLC enabled accurate and reproducible quantitation of analytes in a linear range from 0.03 to 30 μg/mL. After characterizing the peaks in the HPLC-ultraviolet chromatogram obtained from a medicated patient by LC-tandem mass spectrometry, we applied this method to HCC patients taking sorafenib, showing large inter-individual differences in the pharmacokinetic profile. In conclusion, our assay system should be useful for follow-up of patients taking sorafenib and for exploring the association between the pharmacokinetics of sorafenib and its N-oxide and the adverse effects or therapeutic efficacy. PMID:24872323

  18. PPARγ mediates the effects of WIN55,212-2, an synthetic cannabinoid, on the proliferation and apoptosis of the BEL-7402 hepatocarcinoma cells.

    PubMed

    Hong, Yuehui; Zhou, Yuting; Wang, Ying; Xiao, Shunhua; Liao, D Joshua; Zhao, Qing

    2013-11-01

    Cannabis sativa has long been used as a traditional medicine in China. Among its effective compounds are cannabinoids. This study determined the effect of WIN55,212-2 (WIN), a synthetic cannabinoid, on the BEL-7402 human hepatocellular carcinoma (HCC) cell line. The results showed that WIN could decrease the proliferation of BEL-7402 cells. Moreover, WIN could cause apoptosis of the cells via up-regulation of Bax expression, down-regulation of Bcl-2 expression, induction of the mitochondrial membrane potential, increase of caspase-3, -8 and -9 activities, and induction of the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP). The WIN-induced apoptosis was accompanied by the up-regulation of PPARγ expression, the activation of PPARγ DNA binding activity, and a down-regulation of PPARγ target oncogene c-myc. Conversely, the effects of WIN could be attenuated by PPARγ antagonist GW9662, and the WIN induced PPARγ expression was partially attenuated by AM630, a cannabinoid receptor-2 antagonist, whereas the WIN-induced reduction of c-myc expression was partially restored by GW9662. Collectively, our results suggest that WIN can decrease the proliferation and cause apoptosis of the BEL-7402 cells via a mitochondrial-caspase pathway and mediated by PPARγ. These results may provide a basis for the application of WIN in HCC treatment. PMID:24062073

  19. Chemotherapy resistance and metastasis-promoting effects of thyroid hormone in hepatocarcinoma cells are mediated by suppression of FoxO1 and Bim pathway.

    PubMed

    Chi, Hsiang-Cheng; Chen, Shen-Liang; Cheng, Yi-Hung; Lin, Tzu-Kang; Tsai, Chung-Ying; Tsai, Ming-Ming; Lin, Yang-Hsiang; Huang, Ya-Hui; Lin, Kwang-Huei

    2016-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and systemic chemotherapy is the major treatment strategy for late-stage HCC patients. Poor prognosis following chemotherapy is the general outcome owing to recurrent resistance. Recent studies have suggested that in addition to cytotoxic effects on tumor cells, chemotherapy can induce an alternative cascade that supports tumor growth and metastasis. In the present investigation, we showed that thyroid hormone (TH), a potent hormone-mediating cellular differentiation and metabolism, acts as an antiapoptosis factor upon challenge of thyroid hormone receptor (TR)-expressing HCC cells with cancer therapy drugs, including cisplatin, doxorubicin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TH/TR signaling promoted chemotherapy resistance through negatively regulating the pro-apoptotic protein, Bim, resulting in doxorubicin-induced metastasis of chemotherapy-resistant HCC cells. Ectopic expression of Bim in hepatoma cells challenged with chemotherapeutic drugs abolished TH/TR-triggered apoptosis resistance and metastasis. Furthermore, Bim expression was directly transactivated by Forkhead box protein O1 (FoxO1), which was negatively regulated by TH/TR. TH/TR suppressed FoxO1 activity through both transcriptional downregulation and nuclear exclusion of FoxO1 triggered by Akt-mediated phosphorylation. Ectopic expression of the constitutively active FoxO1 mutant, FoxO1-AAA, but not FoxO1-wt, diminished the suppressive effect of TH/TR on Bim. Our findings collectively suggest that expression of Bim is mediated by FoxO1 and indirectly downregulated by TH/TR, leading to chemotherapy resistance and doxorubicin-promoted metastasis of hepatoma cells. PMID:27490929

  20. The novel dual PI3K/mTOR inhibitor NVP-BGT226 displays cytotoxic activity in both normoxic and hypoxic hepatocarcinoma cells

    PubMed Central

    Martelli, Alberto M.; Zauli, Giorgio; Alameen, Ayman A.M.; Ultimo, Simona; Tabellini, Giovanna; McCubrey, James A.; Capitani, Silvano; Neri, Luca M.

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most common lethal human malignancies worldwide and its advanced status is frequently resistant to conventional chemotherapeutic agents and radiation. We evaluated the cytotoxic effect of the orally bioavailable dual PI3K/mTOR inhibitor, NVP-BGT226, on a panel of HCC cell lines, since hyperactivated PI3K/Akt/mTOR signaling pathway could represent a biomolecular target for Small Inhibitor Molecules in this neoplasia. We analyzed the drug activity in both normoxia and hypoxia conditions, the latter playing often a relevant role in the induction of chemoresistance and angiogenesis. In normoxia NVP-BGT226 caused cell cycle arrest in the G0/G1 phase of the cell cycle, induced apoptosis and autophagy at low concentrations. Interestingly the drug inactivated p-Akt and p-S6 at < 10 nM concentration. In hypoxia NVP-BGT226 maintained its cytotoxic efficacy at the same concentration as documented by MTT assays and Western blot analysis. Moreover, the drug showed in hypoxia inhibitory properties against angiogenesis by lowering the expression of the transcription factor HIF-1α and of VEGF. Our results indicate that NVP-BGT226 has a potent cytotoxic effect on HCC cell lines also in hypoxia condition, thus emerging as a potential candidate for cancer treatment in HCC targeted therapy. PMID:26003166

  1. Mast cells mobilize myeloid-derived suppressor cells and Treg cells in tumor microenvironment via IL-17 pathway in murine hepatocarcinoma model.

    PubMed

    Yang, Zhuoshun; Zhang, Biao; Li, Dapeng; Lv, Meng; Huang, Chunmei; Shen, Guan-Xin; Huang, Bo

    2010-01-01

    Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy. PMID:20111717

  2. Antiproliferative Activities of Hot Water Extracts from Culinary-Medicinal Mushrooms, Ganoderma tsugae and Agrocybe cylindracea (Higher Basidiomycetes) on Cancer Cells.

    PubMed

    Chien, Rao-Chi; Tsai, Shu-Yao; Lai, Eric Yih-Cherng; Mau, Jeng-Leun

    2015-01-01

    Using anticancer agents to progress chemotherapy to inhibit the proliferation of cancer cells is an effective means. Two medicinal mushrooms, Ganoderma tsugae and Agrocybe cylindracea, exhibited various physiological effects, and the antiproliferation effect on HL-60, Hep 3B, and C6 cells was studied. The viability of different cancer cells was decreased significantly by hot water extracts from different forms of G. tsugae and A. cylindracea. The hot water extracts from the fruit body, mycelium, and filtrate of A. cylindracea were less effective in inhibiting the antiproliferation of C6, Hep 3B, and HL-60 cells than were those from G. tsugae, as evidenced by their IC50 values. The IC50 values of G. tsugae on C6, Hep 3B, and HL-60 cells were 1.13, 2.73, and 2.60 mg/mL, respectively, whereas those of baby G. tsugae were 1.87, 2.63, and 3.12 mg/mL, respectively. In addition, the filtrates of G. tsugae on C6 and Hep 3B cells were 2.81 and 2.80 mg/mL, respectively. The morphological transformation of 3 cancer cells was observed clearly, and the possible mechanism would be necrosis, apoptosis, or differentiation. Owing to the noticeable effect on antiproliferation of hot water extracts, especially those from G. tsugae, the extract could be of great potential to be used as an alternative cancer therapy. PMID:26082984

  3. Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53.

    PubMed

    Liu, Wen-Ting; Jing, Ying-Ying; Yu, Guo-Feng; Chen, Hong; Han, Zhi-Peng; Yu, Dan-Dan; Fan, Qing-Min; Ye, Fei; Li, Rong; Gao, Lu; Zhao, Qiu-Dong; Wu, Meng-Chao; Wei, Li-Xin

    2016-04-01

    The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail. PMID:27077227

  4. Isoniazid prevents Nrf2 translocation by inhibiting ERK1 phosphorylation and induces oxidative stress and apoptosis

    PubMed Central

    Verma, Ajeet Kumar; Yadav, Arti; Dewangan, Jayant; Singh, Sarvendra Vikram; Mishra, Manisha; Singh, Pradhyumna Kumar; Rath, Srikanta Kumar

    2015-01-01

    Isoniazid is used either alone or in combination with other drugs for the treatment of tuberculosis. It is also used for the prevention of tuberculosis. Chronic treatment of Isoniazid may cause severe liver damage leading to acute liver failure. The mechanism through which Isoniazid causes liver damage is investigated. Isoniazid treatment generates reactive oxygen species and induces apoptosis in Hep3B cells. It induces antioxidative and apoptotic genes leading to increase in mRNA expression and protein levels in Hep3B cells. Whole genome expression analysis of Hep3B cells treated with Isoniazid has resulted in differential expression of various genes playing prime role in regulation of apoptotic, antioxidative, DNA damage, cell signaling, cell proliferation and differentiation pathways. Isoniazid increased cytosolic Nrf2 protein level while decreased nuclear Nrf2 protein level. It also decreased ERK1 phosphorylation and treatment of Hep3B cells with ERK inhibitor followed by Isoniazid resulting in increased apoptosis in these cells. Two dimensional gel electrophoresis results have also shown differential expression of various protein species including heat shock proteins, proteins playing important role in oxidative stress, DNA damage, apoptosis, cell proliferation and differentiation. Results suggest that Isoniazid induces apoptosis through oxidative stress and also prevents Nrf2 translocation into the nucleus by reducing ERK1 phosphorylation thus preventing cytoprotective effect. PMID:26202867

  5. APOBEC3G exerts tumor suppressive effects in human hepatocellular carcinoma.

    PubMed

    Chang, Li-Ching; Kuo, Ting-Yin; Liu, Ching-Wen; Chen, Yaw-Sen; Lin, Hsi-Hsun; Wu, Pei-Fung

    2014-04-01

    In this study, we collected 44 hepatitis B virus surface antigen positivity HBsAg (+) tumor and nontumor hepatocellular tissues from hepatocellular carcinoma (HCC) patients during hepatectomy, and quantified the APOBEC3G (A3G) mRNA by using a real-time PCR. Our results showed higher expression of A3G mRNA in the nontumor tissues than in the tumor tissues of the HBsAg (+) HCC patients. To further investigate this phenomenon, we constructed a pLV-A3G vector and transfected it into the human HCC cell line, Hep 3B. The results of an immunofluorescence analysis showed the overexpression of A3G in the cytoplasm. We then evaluated A3G cytotoxicity by using a cell viability assay (MTS assay), the results of which showed that Hep 3B cell viability was 88 and 58% after the transfection of pLV and pLV-A3G, respectively, indicating the growth inhibitory effects of A3G on Hep 3B cells. To further evaluate the tumor suppressive effects of A3G, we used a plastic pipette tip to scratch Hep 3B cells grown on a culture dish (to 70-80% confluence) after transfection with pLV-A3G. Our data indicated a ratio of wound closure of 100% in the control cells and in the pLV-expressing cells, compared with 43% in the pLV-A3G-overexpressing cells, 72 h after the wound scratch, as observed using phase-contrast microscopy. These results indicated that A3G inhibits wound healing in Hep 3B cells. Overall, our results suggest that A3G inhibits the growth of human hepatoma cells. PMID:24500029

  6. Permissiveness of human hepatoma cell lines for HCV infection

    PubMed Central

    2012-01-01

    Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). Results We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection. PMID:22273112

  7. Clarification of the phenotypic characteristics and anti-tumor activity of Hedyotis diffusa.

    PubMed

    Lee, Hong-Zin; Bau, Da-Tian; Kuo, Chao-Lin; Tsai, Ru-Yin; Chen, Yu-Chang; Chang, Yu-Hao

    2011-01-01

    Hedyotis diffusa Willd. (Rubiaceae) is an important folk herb used to prevent and cure hepatitis and liver cancer in Taiwan. For differentiation of H. diffusa from counterfeits, macroscopic and microscopic characters of H. diffusa, H. corymbosa and H. tenelliflora were examined in this study. According to Trypan blue exclusion assay and Western blot analysis, H. diffusa had a significant inhibition of cell growth and induction of cell apoptosis in COLO 205 (colon cancer), Hep 3B (hepatocellular carcinoma) and H460 (lung cancer) cell lines. This study also used high-performance liquid chromatography (HPLC) to determine the quality control of H. diffusa. The HPLC data showed that ursolic and oleanolic acid are the components of the H. diffusa, consisting of approximately 4.66-4.80% and 1.86-1.96%, respectively. Our study also demonstrated that ursolic acid has significant anti-tumor activity in COLO 205, Hep 3B and H460 cancer cells. PMID:21213409

  8. Hepatitis C virus core protein enhances hepatocellular carcinoma cells to be susceptible to oncolytic vesicular stomatitis virus through down-regulation of HDAC4.

    PubMed

    Moon, Jeong; Kaowinn, Sirichat; Cho, Il-Rae; Min, Do Sik; Myung, Heejoon; Oh, Sangtaek; Kaewpiboon, Chutima; Kraemer, Olive H; Chung, Young-Hwa

    2016-06-01

    Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis. PMID:27150631

  9. ABCG1 is involved in vitamin E efflux.

    PubMed

    Olivier, Maryline; BottG, Remain; Frisdal, Eric; Nowick, Marion; Plengpanich, Wanee; Desmarchelier, Charles; Roi, Stéphanie; Quinn, Carmel M; Gelissen, Ingrid; Jessup, Wendy; Van Eck, Miranda; Guérin, Maryse; Le Goff, Wilfried; Reboul, Emmanuelle

    2014-12-01

    Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcgl-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ- tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macro- phages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux. PMID:25462452

  10. Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Transcription

    NASA Astrophysics Data System (ADS)

    Chandel, N. S.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T.

    1998-09-01

    Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (ρ 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) ρ 0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in ρ 0 cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

  11. Loss of runt-related transcription factor 3 expression leads hepatocellular carcinoma cells to escape apoptosis

    PubMed Central

    2011-01-01

    Background Runt-related transcription factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC). Methods RUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis. Results RUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 ± 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 ± 4% and 4 ± 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation. Conclusion RUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis. PMID:21205319

  12. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  13. Eburicoic Acid, an Active Triterpenoid from the Fruiting Bodies of Basswood Cultivated Antrodia cinnamomea, Induces ER Stress-Mediated Autophagy in Human Hepatoma Cells

    PubMed Central

    Su, Yu-Cheng; Liu, Chun-Ting; Chu, Yung-Lin; Raghu, Rajasekaran; Kuo, Yueh-Hsiung; Sheen, Lee-Yan

    2012-01-01

    Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, can manipulate biological activities, including hepatoprotection, anti-inflammation, anti-hepatitis B virus activity, anticancer activity, etc. In this study, the anti-liver cancer activity and molecular mechanisms of eburicoic acid, the second most abundant triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea was investigated using the human hepatoma Hep 3B cells. The results show that eburicoic acid effectively reduced Hep 3B cell viability within 24 hours, and the IC50 was 18.4 μM, which was equivalent to 8.7 μg/mL. Besides, eburicoic acid induced conversion of LC3-I to LC3-II and a large number of autophagosomes/autolysosomes formation. In depth investigation for the molecular mechanisms, revealed that eburicoic acid firstly promoted reactive oxygen species generation and ATP depletion, leading to endoplasmic reticulum stress, followed by elevated cytosolic calcium ion concentration and BiP expression, downregulated phosphorylation of DAPK, upregulated phosphorylation of Beclin-1, JNK, and Bcl-2, and finally induced autophagy in Hep 3B cells. These results indicate that eburicoic acid has significant anti-liver cancer effects and more distinctive mechanisms. PMID:24716146

  14. Antcin K, an Active Triterpenoid from the Fruiting Bodies of Basswood-Cultivated Antrodia cinnamomea, Inhibits Metastasis via Suppression of Integrin-Mediated Adhesion, Migration, and Invasion in Human Hepatoma Cells.

    PubMed

    Huang, Ya-Ling; Chu, Yung-Lin; Ho, Chi-Tang; Chung, Jing-Gung; Lai, Chiao-I; Su, Yu-Cheng; Kuo, Yueh-Hsiung; Sheen, Lee-Yan

    2015-05-13

    Previous research demonstrated that the ethyl acetate extract from Antrodia cinnamomea suppresses the invasive potential of human breast and hepatoma cells, but the effective compounds are not identified. The main bioactive compounds of A. cinnamomea are ergostane-type triterpenoids, and the content of antcin K is the highest. The objective of this study was to evaluate the antimetastatic activity and mechanisms of antcin K purified from the fruiting body of basswood-cultivated A. cinnamomea on human liver cancer Hep 3B cells. The results showed that adhesion, migration, and invasion of Hep 3B cells were effectively inhibited by antcin K within 24 h of treatment. Antcin K not only reduced the protein expression and activity of MMP-2 and MMP-9 but also down-regulated vimentin and up-regulated E-cadherin in Hep 3B cells. In depth investigation for the molecular mechanism revealed that antcin K could reduce the protein expression of integrin β1, β3, α5, and αv and suppress phosphorylation of FAK, Src, PI3K, AKT, MEK, ERK, and JNK. These results suggested that antcin K was able to inhibit the metastasis of human hepatoma cells through suppression of integrin-mediated adhesion, migration, and invasion. Coupled with these findings, antcin K has a good potential to reduce the risk of liver cancer metastasis. PMID:25911944

  15. The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

    PubMed Central

    Galson, D L; Tsuchiya, T; Tendler, D S; Huang, L E; Ren, Y; Ogura, T; Bunn, H F

    1995-01-01

    The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role. PMID

  16. Prostaglandin E2 accelerates invasion by upregulating Snail in hepatocellular carcinoma cells.

    PubMed

    Zhang, Min; Zhang, Hai; Cheng, Shanyu; Zhang, Dengcai; Xu, Yan; Bai, Xiaoming; Xia, Shukai; Zhang, Li; Ma, Juan; Du, Mingzhan; Wang, Yipin; Wang, Jie; Chen, Meng; Leng, Jing

    2014-07-01

    Our previous studies showed that prostaglandin E2 (PGE2) promotes hepatoma cell growth and migration, as well as invasion; however, the precise mechanism remains elusive. Snail and p65 protein levels were detected in human samples with hepatocellular carcinoma (HCC) by immunohistochemistry (IHC) staining. HCC cell lines (Huh-7 and Hep3B) were used for in vitro experiments. PGE2/Akt/NF-κB pathway was investigated in Huh-7 and Hep3B cells after treatment with PGE2, EP4 receptor (EP4R) agonist, Akt inhibitor, and NF-κB inhibitor, respectively, by real-time reverse transcription (RT)-PCR, Western blotting, and immunofluorescence (IF) staining. In vitro cell invasion assay was performed to evaluate the effect of PGE2 on tumor invasiveness. Knockdown of EP4R was carried out in Huh-7 cells through plasmid-based small interfering RNA (siRNA) approach to confirm the regulation of PGE2 on Snail by EP4R. Dual luciferase reporter assay was performed to assess Snail promoter activity in Huh-7 cell after treatment with EP4R agonist. We found that the protein levels of Snail were higher in HCC tissues than those in control and that PGE2 and EP4R agonist treatment significantly increased Snail expression in Huh-7 and Hep3B cells. EP4R agonist also profoundly promoted invasiveness of Huh-7 cells. Knockdown of the EP4R by siRNA completely blocked the PGE2-induced upregulation of Snail expression and reduced invasiveness of Huh-7 cells. We failed to find that EP4R-induced upregulation of Snail was reversed by inhibition of cAMP response element-binding protein (CREB), a canonical downstream target of EP4R. Alternatively, EP4R agonist treatment significantly increased the levels of phosphorylated EGFR and Akt both in Huh-7 and Hep3B cells. AG1478, an EGFR inhibitor, blocked the phosphorylation of Akt. The levels of phosphorylated IκB increased in Huh-7 cells after treatment with EP4R agonist for 30 min. The levels of phosphorylated p65 started to increase in Huh-7 cells treated

  17. The Impact of a Cash Transfer Program on Cognitive Achievement: The "Bono de Desarrollo Humano" of Ecuador

    ERIC Educational Resources Information Center

    Ponce, Juan; Bedi, Arjun S.

    2010-01-01

    Throughout Latin America, conditional cash transfer (CCT) programs play an important role in social policy. These programs aim to influence the accumulation of human capital, as well as reduce poverty. In terms of educational outcomes, a number of impact evaluation studies have shown that such programs have led to an increase in school enrollment,…

  18. Phenolate based metallomacrocyclic xanthate complexes of Co(II)/Cu(II) and their exclusive deployment in [2 : 2] binuclear N,O-Schiff base macrocycle formation and in vitro anticancer studies.

    PubMed

    Singh, Vinay K; Kadu, Rahul; Roy, Hetal; Raghavaiah, Pallepogu; Mobin, Shaikh M

    2016-01-28

    Potassium salts of phenolate based polydentate xanthate ligands 4,4'-bis(2-dithiocarbonatobenzylideneamino)diphenyl ether () and 4,4'-bis(2-dithiocarbonatonaphthylmethylideneamino)diphenyl ether () have been synthesized and characterized, prior to use. The reaction of or with M(OAc)2 in Et3N affords access to a rare series of binuclear metallomacrocyclic xanthate complexes of the type [M2-μ(2)-bis-(κ(2)S,S-xan(1)/xan(2))] () which quickly forms [2 : 2] binuclear N,O-bidentate Schiff base macrocyclic complexes of the type [M2-μ(2)-bis-(κ(2)N,O-L(1)/L(2))] ( = 4,4'-bis(2-hydroxybenzylideneamino)diphenyl ether, = 4,4'-bis(2-hydroxynaphthylmethylidene-amino)diphenyl ether) via evolution of CS2 in solution. The compounds were characterized by microanalysis, relevant spectroscopy (FT-IR, UV-visible), mass spectrometry (ESI-MS), and powder and single crystal XRD techniques. In vitro anticancer activity of all the compounds was evaluated against HEP 3B (hepatoma) and IMR 32 (neuroblastoma) by the MTT assay. Remarkably, the binuclear copper(ii) xanthate complexes were found to be extremely active against both the cell lines (IC50: 8.1 ± 0.8 μM (), 8.8 ± 1.7 μM () against HEP 3B and 1.9 ± 0.3 μM () and 7.3 ± 0.6 μM () against IMR 32) and this projects them as good candidates for potent antitumor agents and the IC50 values confirm their better potency than the reference drug cisplatin. The flow-cytometric density plot illustrates the induction of apoptosis in HEP 3B and IMR 32 cells after treatment with , , , and . PMID:26674056

  19. Exogenous p53 and ASPP2 expression enhances rAdV-TK/GCV-induced death in hepatocellular carcinoma cells lacking functional p53

    PubMed Central

    Guo, Xianghua; Wei, Feili; Yin, Jiming; Zang, Yunjin; Li, Ning; Chen, Dexi

    2016-01-01

    Suicide gene therapy using herpes simplex virus-1 thymidine kinase (HSV-TK) in combination with ganciclovir (GCV) has emerged as a potential new method for treating cancer. We hypothesize that the efficacy of HSV-TK/GCV therapy is at least partially dependent on p53 status in hepatocellular carcinoma (HCC) patients. Using recombinant adenoviral vectors (rAdV), TK, p53, and ASPP2 were overexpressed individually and in combination in Hep3B (p53 null) and HepG2 (p53 wild-type) cell lines and in primary HCC tumor cells. p53 overexpression induced death in Hep3B cells, but not HepG2 cells. ASPP2 overexpression increased rAdV-TK/GCV-induced HepG2 cell death by interacting with endogenous p53. Similarly, ASPP2 reduced survival in rAdV-TK/GCV-treated primary HCC cells expressing p53 wild-type but not a p53 R249S mutant. Mutated p53 was unable to bind to ASPP2, suggesting that the increase in rAdV-TK/GCV-induced cell death resulting from ASPP2 overexpression was dependent on its interaction with p53. Additionally, γ-H2AX foci, ATM phosphorylation, Bax, and p21 expression increased in rAdV-TK/GCV-treated HepG2 cells as compared to Hep3B cells. This suggests that the combined use of HSV-TK, GCV, rAdV-p53 and rAdV-ASPP2 may improve therapeutic efficacy in HCC patients lacking functional p53. PMID:26934443

  20. Reexpression of ARHI inhibits tumor growth and angiogenesis and impairs the mTOR/VEGF pathway in hepatocellular carcinoma

    SciTech Connect

    Zhao, Xiaohai; Li, Jinfeng; Zhuo, Jianxin; Cai, Liuxin

    2010-12-17

    Research highlights: {yields} Reconstitution of ARHI suppresses the growth of HCC xenografts. {yields} ARHI reexpression impairs tumor angiogenesis in vivo. {yields} Inhibition of the mTOR/VEGF signaling by forced expression of ARHI. {yields} Manipulating ARHI may be of therapeutic benefit in treatment of ARHI-negative HCCs. -- Abstract: The Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI) is frequently downregulated in many types of cancer, including hepatocellular carcinoma (HCC). In this study, we sought to explore the therapeutic implications of ARHI reconstitution in the treatment of HCC. We generated stable cell lines overexpressing ARHI in Hep3B and SK-Hep1 cells, both of which lack endogenous ARHI. The effects of ARHI reexpression on tumor growth and angiogenesis were assessed. Given the key role of mammalian target of rapamycin (mTOR) signaling in HCC progression, we also tested whether ARHI overexpression affected the mTOR pathway. Forced expression of ARHI resulted in a significant inhibition of the proliferation of both Hep3B and SK-Hep1 cells compared to control cells (P < 0.01). Cell cycle analysis revealed a G0-G1 arrest induced by ARHI reexpression. Moreover, ARHI reexpression significantly retarded Hep3B xenograft growth in vivo, and caused a marked reduction in tumor angiogenesis assessed by CD31-stained microvessel count. Western blot analysis of the xenografts showed that ARHI overexpression substantially reduced the phosphorylation of two mTOR substrates, S6K1 and 4E-BP1, indicative of an inactivation of the mTOR pathway. Accompanying with the mTOR inactivation, the angiogenic factors, hypoxia-inducible factor 1 alpha and vascular endothelial growth factor, were significantly downregulated. These data highlighted an important role for ARHI in controlling HCC growth and angiogenesis, therefore offering a possible therapeutic strategy against this malignancy.

  1. Inherent and Acquired Resistance to Paclitaxel in Hepatocellular Carcinoma: Molecular Events Involved

    PubMed Central

    Meena, Avtar Singh; Sharma, Aanchal; Kumari, Ratna; Mohammad, Naoshad; Singh, Shivendra Vikram; Bhat, Manoj Kumar

    2013-01-01

    Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and is a major cause of cancer related deaths worldwide. Only 10 to 20% of HCC can be surgically excised. Therefore, chemotherapeutic intervention and treatment is essential for achieving favorable prognosis. However, therapeutic outcome of chemotherapy is generally poor owing to inherent resistance of cancer cells to the treatment or due to development of acquired resistance. To differentiate and delineate the molecular events, we developed drug resistant Hep3B cells (DRC) by treating cells with the increasing concentration of paclitaxel. We also developed a unique single cell clone of Hep3B cells (SCC) by selecting single cell colonies and screening them for resistant phenotype. Interestingly, both DRC and SCC were resistant to paclitaxel in comparison to parental Hep3B cells. We analyzed the contributory factors that may be involved in the development of resistance. As expected, level of P-glycoprotein (P-gp) was elevated in DRC. In addition, Caveolin-1 (Cav-1), Fatty acid synthase (FASN) and Cytochrome P450 (CYP450) protein levels were elevated in DRC whereas in SCC, FASN and CYP450 levels were elevated. Downregulation of these molecules by respective siRNAs and/or by specific pharmacological inhibitors resensitized cells to paclitaxel. Interestingly, these drug resistant cells were also less sensitive to vinblastine, doxorubicin and methotrexate with the exception of cisplatin. Our results suggested that differential levels of P-gp, Cav-1 and FASN play a major role in acquired resistant phenotype whereas FASN level was associated with the presentation of inherent resistant phenotype in HCC. PMID:23613870

  2. In vitro and in vivo cytotoxic effects of PRIMA-1 on hepatocellular carcinoma cells expressing mutant p53ser249.

    PubMed

    Shi, Hong; Lambert, Jeremy M R; Hautefeuille, Agnes; Bykov, Vladimir J N; Wiman, Klas G; Hainaut, Pierre; Caron de Fromentel, Claude

    2008-07-01

    Hepatocellular carcinoma (HCC) is highly lethal due to limited curative options. In high-incidence regions, such as parts of Africa and Southeastern Asia, >50% of cases carry an AGG to AGT mutation at codon 249 of the TP53 gene, considered as a 'signature' of mutagenesis by aflatoxins. The protein product, p53ser249, may represent a therapeutic target for HCC. The small molecule p53 reactivation and induction of massive apoptosis (PRIMA)-1 has been shown to induce apoptosis in tumour cells by reactivating the transactivation capacity of some p53 mutants. In this study, we have investigated the cytotoxic effects of PRIMA-1 on HCC cells expressing p53ser249. In p53-null Hep3B cells, over-expression of p53ser249 or p53gln248 by stable transfection increased the cytotoxicity of PRIMA-1 at 50 muM. Furthermore, PRIMA-1 treatment delayed the growth of p53ser249-expressing Hep3B cells xenografted in severe combined immunodeficiency mice. However, PRIMA-1 did not restore wild-type DNA binding and transactivation activities to p53ser249 or to p53gln248 in Hep3B cells. Moreover, in PLC/PRF/5, a HCC cell line constitutively expressing p53ser249, small interfering RNA (siRNA) silencing of the mutant increased the cytotoxic effect of PRIMA-1. These apparently contradictory effects can be reconciled by proposing that p53ser249 exerts a gain-of-function effect, which favours the survival of HCC cells. Thus, both inhibition of this effect by PRIMA-1 and removal of the mutant by siRNA can lead to the decrease of survival capacity of HCC cells. PMID:18048389

  3. Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells.

    PubMed

    Scandurro, A B; Weldon, C W; Figueroa, Y G; Alam, J; Beckman, B S

    2001-07-01

    The molecular details of hypoxia-induced cellular responses have been difficult to identify since there is as yet no known oxygen receptor. We used cDNA microarray technology to extend our studies pertaining to these molecular details in human hepatocellular carcinoma (Hep3B) cells that produce erythropoietin (Epo) in response to hypoxia. Of approximately 1200 genes in the array, those associated with integrin-linked kinase (ILK), fibronectin precursor and glycogen synthase kinase-3beta (GSK-3beta) were markedly stimulated after exposure of Hep3B cells to low oxygen (1%) for 6 h. Epo, HIF-1, and von Hippel-Lindau cDNAs were measured in parallel as markers of low oxygen responses in Hep3B cells. ILK is a serine, threonine protein kinase that interacts with the cytoplasmic domains of integrin beta1 and beta3. This interaction localizes ILK to focal adhesion plaques. ILK is stimulated by cell-fibronectin interaction as well as insulin. It is regulated in a phosphatidylinositol 3-kinase dependent manner and can phosphorylate protein kinase B (PKB/AKT) and GSK-3beta. As a result of these and other activities ILK has been shown to affect anchorage-independent cell survival, cell cycle progression and tumorigenesis in nude mice. ILK has also been implicated in the Wnt pathway and as a critical target in PTEN-dependent tumor therapies. To our knowledge this is the first report implicating the ILK pathway in low oxygen responses. Other genes identified as a result of the microarray analysis not previously known to change as a result of low oxygen treatment were elongation factor-1alpha, glycyl-tRNA synthetase, and laminin receptor protein-1. These findings were all corroborated by RT-PCR assays and in some instances Western blot analysis. PMID:11408933

  4. Human cell line-dependent WC-Co nanoparticle cytotoxicity and genotoxicity: a key role of ROS production.

    PubMed

    Paget, V; Moche, H; Kortulewski, T; Grall, R; Irbah, L; Nesslany, F; Chevillard, S

    2015-02-01

    Although tungsten carbide-cobalt (WC-Co) nanoparticles (NPs) have been widely used because of their robustness, their risk to human health remains poorly studied, despite the International Agency for Research on Cancer (IARC) classifying them as "probably carcinogenic" for humans (Group 2A) in 2006. Our current study aimed at defining the cytotoxicity and genotoxicity of one set of commercially available 60-nm diameter WC-Co NPs on three human cell lines representative of potential target organs: A549 (lung), Hep3B (liver), and Caki-1 (kidney). The cytotoxicity of WC-Co NPs was determined by evaluating cell impedance (xCELLigence), cell survival/death, and cell cycle checkpoints. Flow cytometry was used to not only evaluate cell cycle checkpoints, but to also estimate reactive oxygen species (ROS) generation. In addition, γ-H2Ax foci detection (confocal microscopy), considered to be the most sensitive technique for studying DNA double-strand breaks, was utilized to evaluate genotoxicity. As a final part of this study, we assessed the cellular incorporation of WC-Co NPs, first byflow cytometry (side scatter), and then by confocal microscopy (light reflection) to ensure that the NPs had entered cells. Overall, our current findings demonstrate that WC-Co NPs induce cell mortality, DNA double-strand breaks, and cell cycle arrest in human renal (Caki-1) and liver (Hep3B) cell lines, but do not induce significant cytotoxic effects in A549 lung cells. Interestingly, although WC-Co NPs effectively entered the cells in all 3 lines tested, ROS were detected in Caki-1 and Hep3B, but not in A549. This may explain the great differences in the cytotoxic and genotoxic effects we observed between these lines. PMID:25398624

  5. Novel histone deacetylase inhibitor MPT0G009 induces cell apoptosis and synergistic anticancer activity with tumor necrosis factor-related apoptosis-inducing ligand against human hepatocellular carcinoma.

    PubMed

    Chen, Mei-Chuan; Huang, Hui-Hsuan; Lai, Chin-Yu; Lin, Yi-Jyun; Liou, Jing-Ping; Lai, Mei-Jung; Li, Yu-Hsuan; Teng, Che-Ming; Yang, Chia-Ron

    2016-01-01

    Hepatocellular carcinoma (HCC) is a frequent cause of cancer-related death; therefore, more effective anticancer therapies for the treatment of HCC are needed. Histone deacetylase (HDAC) inhibitors serve as promising anticancer drugs because they can induce cell growth arrest and apoptosis. We previously reported that 3-[1-(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-5-yl]-N-hydroxyacrylamide (MPT0G009)-a novel 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines compound-demonstrated potent pan-HDAC inhibition and anti-inflammatory effects. In this study, we evaluated the anti-HCC activity of MPT0G009 in vitro and in vivo. Growth inhibition, apoptosis, and inhibited HDAC activity induced by MPT0G009 were more potent than a marketed HDAC inhibitor SAHA (Vorinostat). Furthermore, MPT0G009-induced apoptosis of Hep3B cells was characterized by an increase in apoptotic (sub-G1) population, loss of mitochondrial membrane potential, activation of caspase cascade, increased levels of pro-apoptotic protein (Bim), and decreased levels of anti-apoptotic proteins (Bcl-2, Bcl-xL, and FLICE-inhibitory protein); the downregulation FLIP by MPT0G009 is mediated through proteasome-mediated degradation and transcriptional suppression. In addition, combinations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with lower concentrations (0.1 μM) of MPT0G009 were synergistic in cell growth inhibition and apoptosis in HCC cells. In the in vivo model, MPT0G009 markedly reduced Hep3B xenograft tumor volume, inhibited HDAC activities, and induced apoptosis in the Hep3B xenografts. Our results demonstrate that MPT0G009 is a potential new candidate drug for HCC therapy. PMID:26587975

  6. MicroRNA-215 is upregulated by treatment with Adriamycin and leads to the chemoresistance of hepatocellular carcinoma cells and tissues.

    PubMed

    Wang, Li; Wang, Yan Ming; Xu, Song; Wang, Wei Guo; Chen, Yang; Mao, Jing Yu; Tian, Bo Le

    2015-10-01

    Non-coding microRNAs (miRNAs), involved in post-transcriptional control, are widely involved in the mechanism of cellular resistance to antitumor chemotherapy. Ectopic expression of one of these miRNAs, miRNA‑215 (miR‑215), leads to chemoresistance by directly targeting dihydrofolate reductase (DHFR) and thymidylate synthase (TS), which are two of the most important targets of chemotherapeutic agents. This indicates the possible upregulation of endogenous miR‑215 in the process of chemoresistance by interfering with important transcripts. In the present study, the upregulation of miR‑215 was examined in hepatocellular carcinoma (HCC) subcell lines, Adriamycin (ADM)‑resistant HepG2 (HepG2/AR), Hep3B (Hep3B/AR) cell lines, and in ADM‑treated patients with HCC. Upregulated miR‑215 directly targeted DHFR and TS mRNA and reduced their protein expression levels, without altering mRNA levels. The ectopic expression of miR‑215 anti‑sense oligo‑nucleotides in HepG2/AR and Hep3B/AR cells enhanced chemosensitivity, whereas the expression of the miR‑215 mimics led to chemoresistance. Notably, the upregulation of miR‑215 indirectly increased the protein levels of P53 and P21 levels in the HepG2 cells, which contain functional P53, which is expected to result in the inhibition of proliferation and colony formation. Taken together, the present study demonstrated that the upregulation of miR‑215 resulting from ADM treatment in HCC cells leads to the development of insensitivity to ADM and worsens the prognosis of patients with HCC exhibiting mutated P53. PMID:26135967

  7. Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells.

    PubMed

    Lai, Chiao-I; Chu, Yung-Lin; Ho, Chi-Tang; Su, Yu-Cheng; Kuo, Yueh-Hsiung; Sheen, Lee-Yan

    2016-01-01

    Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy. PMID:26870680

  8. Novel histone deacetylase inhibitor MPT0G009 induces cell apoptosis and synergistic anticancer activity with tumor necrosis factor-related apoptosis-inducing ligand against human hepatocellular carcinoma

    PubMed Central

    Lai, Chin-Yu; Lin, Yi-Jyun; Liou, Jing-Ping; Lai, Mei-Jung; Li, Yu-Hsuan; Teng, Che-Ming; Yang, Chia-Ron

    2016-01-01

    Hepatocellular carcinoma (HCC) is a frequent cause of cancer-related death; therefore, more effective anticancer therapies for the treatment of HCC are needed. Histone deacetylase (HDAC) inhibitors serve as promising anticancer drugs because they can induce cell growth arrest and apoptosis. We previously reported that 3-[1-(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-5-yl]-N-hydroxyacrylamide (MPT0G009)—a novel 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines compound—demonstrated potent pan-HDAC inhibition and anti-inflammatory effects. In this study, we evaluated the anti-HCC activity of MPT0G009 in vitro and in vivo. Growth inhibition, apoptosis, and inhibited HDAC activity induced by MPT0G009 were more potent than a marketed HDAC inhibitor SAHA (Vorinostat). Furthermore, MPT0G009-induced apoptosis of Hep3B cells was characterized by an increase in apoptotic (sub-G1) population, loss of mitochondrial membrane potential, activation of caspase cascade, increased levels of pro-apoptotic protein (Bim), and decreased levels of anti-apoptotic proteins (Bcl-2, Bcl-xL, and FLICE-inhibitory protein); the downregulation FLIP by MPT0G009 is mediated through proteasome-mediated degradation and transcriptional suppression. In addition, combinations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with lower concentrations (0.1 μM) of MPT0G009 were synergistic in cell growth inhibition and apoptosis in HCC cells. In the in vivo model, MPT0G009 markedly reduced Hep3B xenograft tumor volume, inhibited HDAC activities, and induced apoptosis in the Hep3B xenografts. Our results demonstrate that MPT0G009 is a potential new candidate drug for HCC therapy. PMID:26587975

  9. A new phenyl glycoside from the aerial parts of Equisetum hyemale.

    PubMed

    Jin, Mei; Zhang, Changhao; Zheng, Tie; Yao, Dalei; Shen, Le; Luo, Jie; Jiang, Zhe; Ma, Juan; Jin, Xue-Jun; Cui, Jiongmo; Lee, Jung Joon; Li, Gao

    2014-01-01

    A new phenyl glycoside, 2-(sophorosyl)-1-(4-hydroxyphenyl)ethanone (9), was isolated from the ethanolic extract of the aerial parts of Equisetum hyemale L., together with eight known compounds (1-8). The structures of these compounds were elucidated using a combination of spectroscopic analyses and chemical method. Of these nine compounds, 4 and 7 showed hepatoprotective effects towards tacrine-induced cytotoxicity in Hep 3B cells with EC50 values of 42.7 ± 1.5 and 132.6 ± 2.8 μM, respectively. PMID:25117054

  10. Development of a cell viability assay to assess drug metabolite structure-toxicity relationships.

    PubMed

    Rana, Payal; Will, Yvonne; Nadanaciva, Sashi; Jones, Lyn H

    2016-08-15

    Many adverse drug reactions are caused by the cytochrome P450 (CYP)-dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-induced toxicity and to improve the safety of drug candidates, we developed a simple cell viability assay by combining a bioactivation system (human CYP3A4, CYP2D6 and CYP2C9) with Hep3B cells. We screened a series of drugs to explore structural motifs that may be responsible for CYP450-dependent activation caused by reactive metabolite formation, which highlighted specific liabilities regarding certain phenols and anilines. PMID:27397500

  11. Effects of 5-azacytidine and butyrate on differentiation and apoptosis of hepatic cancer cell lines.

    PubMed Central

    Wang, X M; Wang, X; Li, J; Evers, B M

    1998-01-01

    OBJECTIVE: To determine the cellular effects of 5-azacytidine (5-azaC) and sodium butyrate on two human liver cancers, HepG2 and Hep3B. SUMMARY BACKGROUND DATA: Primary liver cancer is a significant health problem; treatment options are limited and prognosis is poor. Recent studies have focused on the role that programmed cell death (i.e., apoptosis) plays in both normal and neoplastic growth: certain genes can either suppress (e.g., Bcl-2, Bcl-xL) or promote (e.g., Bik, Bax, Bak) apoptosis. The identification of novel agents targeted to specific molecular pathways may be beneficial in the treatment of this disease. METHODS: Human liver cancer cell lines HepG2 and Hep3B were treated with 5-azaC alone, butyrate alone, or 5-azaC and butyrate. Morphologic and proliferative changes were assessed by light microscopy and 5-bromo-2'-deoxyuridine staining; flow cytometry was used to determine cell cycle characteristics. Apoptosis was assessed by DNA laddering and the in situ apoptosis detection assay using the TdT-mediated dUTP nick end labeling method. In addition, total RNA and protein were analyzed by ribonuclease protection and Western blot, respectively, to assess changes in the expression of apoptosis-related genes. RESULTS: Treatment with either 5-azaC or butyrate inhibited cell growth and induced apoptosis in both HepG2 and Hep3B cells; the combination of 5-azaC and butyrate was not more effective than either agent alone. 5-azaC alone resulted in a more differentiated-appearing morphology and G2 cell cycle arrest in both cell lines. Treatment with 5-azaC or butyrate affected the expression levels of proteins of the Bcl-2 family. CONCLUSIONS: Both 5-azaC and butyrate induced apoptosis in the HepG2 and Hep3B liver cancer cells; 5-azaC treatment alone produced G2 arrest in both cell lines. Proteins of the Bcl-2 family may play a role in the cellular changes that occur with treatment, but further studies are required to define this potential role. Products of the

  12. Eudesmane-type sesquiterpenoids from Salvia plebeia inhibit IL-6-induced STAT3 activation.

    PubMed

    Jang, Hyun-Jae; Oh, Hyun-Mee; Hwang, Joo Tae; Kim, Mi-Hwa; Lee, Soyoung; Jung, Kyungsook; Kim, Young-Ho; Lee, Seung Woong; Rho, Mun-Chual

    2016-10-01

    Seven eudesmane-type sesquiterpenoid lactones and the known plebeiolide C were isolated from an ethanol-soluble extract of the aerial parts of Salvia plebeia R. Br. Their structures were determined via NMR and MS, and their absolute configurations were elucidated using ECD, and X-ray crystallographic analysis, as well as the modified Mosher ester method. All isolates were evaluated for their inhibitory effects on IL6-induced STAT3 promoter activation in stably transfected Hep3B cells. Of these isolates, eudebeiolide D exhibited an inhibitory effect with the IC50 value of 1.1 μM. PMID:27506573

  13. Design, Synthesis and Structure-Activity Relationship Studies of Novel 4 (1-adamantyl) Phenyl Analogues as HIF-1α Inhibitors.

    PubMed

    Xia, Yan; Duan, Qiong; Zhao, Bao-Hua; Li, Dong-Feng; Hou, Rui-Bin

    2016-01-01

    Hypoxia inducible factor-1 (HIF-1) is a key mediator during cancer cells to adapt tumor hypoxic condition. In this study, a series of adamantane-based compounds were synthesized and evaluated as potential inhibitors of HIF-1α. Examination of their structure-activity relationship (SAR) identified the adamantane-containing indole derivative 20a as a potent inhibitor of HIF-1α in Hep3B cell lines under tumor hypoxia (IC50 = 0.02 µM). The study herein may provide valuable information for the development of novel therapeutics against cancer and tumor angiogenesis. PMID:26548744

  14. Liquid chromatography with mass spectrometry and NMR spectroscopy based discovery of cytotoxic principles from Daphne tangutica Maxim.

    PubMed

    Yang, Xinzhou; Huang, Mi; Zheng, Sijian; Ma, Xinhua; Wan, Dingrong; Feng, Yunjiang

    2016-06-01

    An ethyl acetate extract from the barks of the ethnic Chinese medicine Daphne tangutica Maxim. exhibited antihepatocellular carcinoma activity against HepG2 and Hep3B cell lines. By using high-performance liquid chromatography based activity profiling in combination with offline liquid chromatography with mass spectrometry and NMR analysis, we rapidly identified ten major components of the extract, including seven active principles, coumarins (1-4) and biscoumarins (7, 8, 10), along with three inactive flavonoids (5, 6, 9). This study demonstrated that our combined protocol can be used as an important strategy for chemical profiling, dereplication as well as the identification of bioactive compounds from herbal medicines. PMID:27062225

  15. Isolation and Characterization of Dammarane-Type Saponins from Gynostemma pentaphyllum and Their Inhibitory Effects on IL-6-Induced STAT3 Activation.

    PubMed

    Lee, Chul; Lee, Jin Woo; Jin, Qinghao; Jang, Hari; Jang, Hyun-Jae; Rho, Mun-Chual; Lee, Myung Koo; Lee, Chong Kil; Lee, Mi Kyeong; Hwang, Bang Yeon

    2015-05-22

    Five new dammarane-type saponins, gypenosides GD1-GD5 (1-5), along with six known saponins (6-11), were isolated from the aerial parts of Gynostemma pentaphyllum using various chromatographic methods. Their structures were elucidated by a combination of spectroscopic and spectrometric data, including 1D and 2D NMR and HRESIMS. All isolates were tested for their inhibitory effects on IL-6-induced STAT3 promoter activity in Hep3B cells. Compounds 1, 9, and 11 displayed potent inhibitory effects, with IC50 values ranging from 0.27 to 0.59 μM. PMID:25895106

  16. Propiconazole increases reactive oxygen species levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450 enzyme mediated process

    EPA Science Inventory

    Propiconazole induces hepatocarcinomas and hepatoadenomas in mice and is a rat liver tumor promoter. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicate that ...

  17. Cold-water extracts of Grifola frondosa and its purified active fraction inhibit hepatocellular carcinoma in vitro and in vivo.

    PubMed

    Lin, Chia-Hung; Chang, Ching-Yao; Lee, Kuan-Rong; Lin, Hui-Ju; Lin, Wu-Chou; Chen, Ter-Hsin; Wan, Lei

    2016-07-01

    Mushrooms are used in traditional Chinese medicine to treat a variety of diseases. Grifola frondosa (GF) is an edible mushroom indigenous to many Asian countries with a large fruiting body characterized by overlapping caps. In particular, GF is known for its anti-tumor activity, which has been targeted by scientific and clinical research. This study aimed to investigate the effects of the cold-water extract of GF (GFW) and its active fraction (GFW-GF) on autophagy and apoptosis, and the underlying mechanisms in vitro and in vivo Our results revealed that GFW and GFW-GF inhibited phosphatidylinositol 3-kinase (PI3K) and stimulated c-Jun N-terminal kinase (JNK) pathways, thereby inducing autophagy. We also demonstrated that GFW and GFW-GF inhibited proliferation, induced cell cycle arrest, and apoptosis in Hep3B hepatoma cells. GFW and GFW-GF markedly arrested cells in S phase and promoted cleavage of caspase-3 and -9. In addition, GFW and GFW-GF decreased the expression levels of the anti-apoptotic proteins protein kinase B and extracellular signal-regulated kinase. We also found that GFW significantly inhibited tumor growth in nude mice implanted with Hep3B cells. Our work demonstrates that GF and its active fraction inhibit hepatoma growth by inducing autophagy and apoptosis. PMID:27013543

  18. Design and Synthesis of 2-(3-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one Analogs as Potent Antitumor Agents that Inhibit Tubulin Assembly

    PubMed Central

    Chang, Yu-Hsun; Hsu, Mei-Hua; Wang, Sheng-Hung; Huang, Li-Jiau; Qian, Keduo; Morris-Natschke, Susan L.; Hamel, Ernest; Kuo, Sheng-Chu; Lee, Kuo-Hsiung

    2009-01-01

    As part of our continuing investigation of azo-flavonoid derivatives as potential anticancer drug candidates, a series of 2-aryl-6,7-methylenedioxyquinolin-4-one analogs was designed and synthesized. The design combined structural features of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (CHM-1), a previously discovered compound with potent in vivo antitumor activity, and 2-arylquinolin-4-ones identified by CoMFA models. The newly synthesized analogs were evaluated for cytotoxicity against seven human cancer cell lines, and structure-activity relationship (SAR) correlations were established. Analogs 1, 37, and 39 showed potent cytotoxicity against different cancer cell lines. Compound 1 demonstrated selective cytotoxicity against Hep 3B (hepatoma) cells. Compound 37 was cytotoxic against HL-60 (leukemia), HCT-116 (colon cancer), Hep 3B (hepatoma), and SK-MEL-5 (melanoma) cells. Compound 39 exhibited broad cytotoxicity against all seven cancer cell lines, with IC50 values between 0.07–0.19 µM. Results from mechanism of action studies revealed that these new quinolone derivatives function as antitubulin agents. PMID:19719238

  19. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    PubMed

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes. PMID:24302965

  20. Gold Nanoparticles and Radiofrequency in Experimental Models for Hepatocellular Carcinoma

    PubMed Central

    Raoof, Mustafa; Corr, Stuart J.; Zhu, Cihui; Cisneros, Brandon T.; Kaluarachchi, Warna D; Phounsavath, Sophia; Wilson, Lon J.; Curley, Steven A.

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most lethal and chemo-refractory cancers, clearly, alternative treatment strategies are needed. We utilized 10nm gold nanoparticles as a scaffold to synthesize nanoconjugates bearing a targeting antibody (cetuximab, C225) and gemcitabine. Loading efficiency of gemcitabine on the gold nanoconjugates was 30%. Targeted gold nanoconjugates in combination with RF were selectively cytotoxic to EGFR expressing Hep3B and SNU449 cells when compared to isotype particles with/without RF (p<0.05). In animal experiments, targeted gold nanoconjugates halted the growth of subcutaneous Hep3B xenografts in combination with RF exposure (p<0.05). These xenografts also demonstrated increased apoptosis, necrosis and decreased proliferation compared to controls. Normal tissues were unharmed. We have demonstrated that non-invasive RF-induced hyperthermia when combined with targeted delivery of gemcitabine is more effective and safe at dosages ~275-fold lower than the current clinically-delivered systemic dose of gemcitabine. PMID:24650884

  1. SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells.

    PubMed

    Turkoglu, Sumeyye Aydogan; Kockar, Feray

    2016-01-01

    ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition. PMID:26299656

  2. Steroids from the rhizome of Anemarrhena asphodeloides and their cytotoxic activities.

    PubMed

    Sun, Yu; Wu, Jie; Sun, Xue; Huang, Xiaoxiao; Li, Lingzhi; Liu, Qingbo; Song, ShaoJiang

    2016-07-01

    Cancer remains a major killer worldwide. To search for novel naturally occurring compounds that are cytotoxic to cancer cells to be used as lead structures for drug development, five new steroids (1-5) along with seven known ones (6-12) were isolated from the rhizome of Anemarrhena asphodeloides Bge. Their structures were established by detailed spectral studies, including 1D-NMR, 2D-NMR, HR-ESI-MS and by comparison with literature data. These compounds exhibited different levels of growth inhibition against A549, HepG2, Hep3B, Bcap37 and MCF7 cell lines in vitro. Compounds 9, 10 and 11 showed potent inhibitory against all the tested cell lines with IC50 values ranging from 0.35±0.15 to 25.53±0.31μM. The three compounds displayed stronger inhibitory activities against A549, HepG2 and Hep3B cell lines compared with the positive control 5-fluorouracil. The experimental data obtained permit us to identify the roles of the sugar moieties, hydroxyl group, double bond and F-ring with regard to their cytotoxic activities. PMID:27189676

  3. Overexpression of α1,6-fucosyltransferase in hepatoma enhances expression of Golgi phosphoprotein 2 in a fucosylation-independent manner.

    PubMed

    Kawamoto, Sayuri; Moriwaki, Kenta; Nakagawa, Tsutomu; Terao, Mika; Shinzaki, Shinichiro; Yamane-Ohnuki, Naoko; Satoh, Mitsuo; Mehta, Anand S; Block, Timothy M; Miyoshi, Eiji

    2011-07-01

    Golgi phosphoprotein 2 (GP73) is a type II Golgi protein, which was found on examination of the fucosylated proteome as a potential tumor marker for hepatocellular carcinoma (HCC). The serum levels of both total and fucosylated GP73 were increased in the sera of patients with HCC. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and is catalyzed by α1,6-fucosyltransferase (Fut8). In the present study, we investigated the effect of Fut8 overexpression on GP73 production in the human hepatoma cell line Hep3B. The Fut8 expression vector was transfected into Hep3B cells and the expression of GP73 was investigated by Western blotting and real-time PCR. Overexpression of Fut8 dramatically enhanced the expression of GP73 at the transcriptional level. Surprisingly, this effect was not dependent on cellular fucosylation. Overexpression of a mutant Fut8, which was unable to be localized to the Golgi, did not induce GP73 production, suggesting that the localization of Fut8 in the Golgi apparatus was important for the increase in GP73 expression. This is the first demonstration of GP73 regulation through overexpression of a glycosyltransferase, which may lead to Golgi stress. PMID:21503570

  4. Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

    PubMed

    Iqbal Ahmed, C M; Johnson, D E; Demers, G W; Engler, H; Howe, J A; Wills, K N; Wen, S F; Shinoda, J; Beltran, J; Nodelman, M; Machemer, T; Maneval, D C; Nagabhushan, T L; Sugarman, B J

    2001-10-01

    A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells. PMID:11687902

  5. Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.

    PubMed Central

    Blanchard, K L; Acquaviva, A M; Galson, D L; Bunn, H F

    1992-01-01

    Transcription of the human erythropoietin (Epo) gene is stimulated by exposure to hypoxia and/or cobalt in whole animals and in Hep3B cells. We have systematically investigated the promoter and 3' enhancer elements necessary for this induction by transient transfection of Hep3B cells. We define a promoter region of 53 bp and an enhancer region of 43 bp that confer hypoxia and cobalt inducibility. Each element gives rise to a 6- to 10-fold induction alone. In combination they produce a 50-fold induction after stimulation, similar to the 50- to 100-fold induction of the endogenous Epo gene. Two areas of DNA sequence homology are present in these regions. We demonstrate specific DNA-protein interactions in the enhancer and the ability of the promoter element to compete with these interactions in electrophoretic mobility shift assays. DNase I footprinting and methylation interference data further refine the cis-acting element in the 43-bp enhancer to a short region containing a direct repeat of a steroid/thyroid hormone receptor response element half-site separated by a 2-bp gap. Two half-site consensus sequences are also present in the 53-bp promoter. Site-specific mutation of the half-site sequences in the enhancer destroys the functional activity of the enhancer. Images PMID:1448072

  6. Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells

    PubMed Central

    Youn, Myung-Ja; Kim, Jin-Kyung; Park, Seong-Yeol; Kim, Yunha; Kim, Se-Jin; Lee, Jin Seok; Chai, Kyu Yun; Kim, Hye-Jung; Cui, Ming-Xun; So, Hong Seob; Kim, Ki-Young; Park, Raekil

    2008-01-01

    AIM: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines, HepG2 and Hep3B cells. METHODS: The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS: HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression. CONCLUSION: Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma. PMID:18203281

  7. Dexamethasone impairs hypoxia-inducible factor-1 function

    SciTech Connect

    Wagner, A.E.; Huck, G.; Stiehl, D.P.; Jelkmann, W.; Hellwig-Buergel, T.

    2008-07-25

    Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of {alpha}- and {beta}-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1{alpha} levels in the cytosol of HepG2 cells, while nuclear HIF-1{alpha} levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

  8. N-all-trans-retinoyl-L-proline inhibits metastatic potential of hepatocellular carcinoma cells.

    PubMed

    Wu, Xing Zhong; Shi, Peng-Chen; Hu, Ping; Chen, Yi; Ding, Sheng-Song

    2006-08-01

    Tumor metastasis is usually a serious problem in tumor patients because of the lack of therapeutic approaches. A new compound, N-all-trans-retinoyl-L-proline (ATRP), has been developed and its metastasis inhibition activity has been studied. Low concentrations of ATRP have already been found to inhibit hepatocellular carcinoma cells (HCC) in a dose- and time-dependent manner by inducing the expression of p27(kip). We found that ATRP inhibited metastasis-associated behaviors in Hep3B cells, such as cell migration, invasion, collagen adhesion and gelatinase expression, more significantly than retinoic acid. Further, such inhibitory activities were observed in the regulation of cellular surface fucosylated epitope functions, such as binding of ulex europaeus lectin, expression of Lewis x, y and b, and activity of alpha1,3 fucosyltransferase. Hep3B cells pretreated with ATRP showed a significantly reduced incidence of experimental intrahepatic metastasis in nude mice. We conclude that ATRP is an alternative inhibitor and potential therapeutic agent for HCC metastasis with a different mechanism of action from ATRP. PMID:16806999

  9. Hydroxyethyl starch-10-hydroxy camptothecin conjugate: synthesis, pharmacokinetics, cytotoxicity and pharmacodynamics research.

    PubMed

    Li, Guofei; Cai, Cuifang; Qi, Yingjie; Tang, Xing

    2016-01-01

    10-hydroxy camptothecin (10-HCPT) is an antitumor agent effective in the treatment of several solid tumors but its use is hampered by poor water solubility, low lactone stability, short plasma half-life and dose-limiting toxicity. In this study, 10-HCPT-hydroxyethyl starch (HES) conjugate was prepared to overcome these limits of 10-HCPT. The solubility of 10-HCPT conjugate was 0.72 mg/ml, about 100 times to free 10-HCPT. The 10-HCPT conjugate showed good sustained release effect in phosphate-buffered saline (PBS), rat plasma and liver homogenate. Meanwhile, 10-HCPT-HES conjugate achieved much lower IC50 and higher cytotoxicity effects than the free 10-HCPT on Hep-3B liver cancer cells. The pharmacokinetics results of 10-HCPT-HES conjugate demonstrated that the biological half-life of 10-HCPT was increased from 10 min to 4.38 h and the bioavailability was 40 times higher than the commercial 10-HCPT injection. The pharmacodynamics results indicated that 10-HCPT-HES conjugate had a better antitumor efficiency against nude mouse with Hep-3B tumor than the commercial 10-HCPT injection, and the inhibition ratio of tumor was 83.9 and 27.8%, respectively, at the same administration dosage. These findings suggest that 10-HCPT-HES conjugate is a promising drug delivery system providing improved long circulating effect, greater stability and better antitumor effect. PMID:24833272

  10. Hydroxyethyl starch conjugates for improving the stability, pharmacokinetic behavior and antitumor activity of 10-hydroxy camptothecin.

    PubMed

    Li, Guofei; Li, Yan; Tang, Yilin; Zhang, Yu; Zhang, Yan; Yin, Tian; Xu, Hui; Cai, Cuifang; Tang, Xing

    2014-08-25

    10-Hydroxy camptothecin (10-HCPT)-hydroxyethyl starch (HES) conjugates were prepared to improve the water solubility, prolong the half-life in plasma and increase the antitumor efficacy of 10-HCPT, and the structures of the conjugates were confirmed by NMR and infrared spectroscopy. The 10-HCPT conjugates showed good sustained release effect in phosphate-buffered saline (PBS), rat plasma and liver homogenate. Meanwhile, 10-HCPT-HES conjugates achieved much lower IC50 and higher cytotoxicity effects than the free 10-HCPT on Hep-3B and SMMC-7721 cell lines. The pharmacokinetics results of 10-HCPT-HES conjugates demonstrated that the biological half-life of 10-HCPT was increased from 10 min to 2.94 h and 3.76 h, respectively, in comparison with the commercial 10-HCPT injection. The pharmacodynamics results indicated that 10-HCPT-HES conjugate had a better antitumor efficiency against nude mouse with Hep-3B tumor than the commercial 10-HCPT injection, and the inhibition ratio of tumor was 78.3% and 31.5%, respectively, at the dose of 1.0 mg/kg. These findings suggest that 10-HCPT-HES conjugate is a promising drug delivery system providing improved long circulating effect, greater stability and better antitumor effect. PMID:24861941

  11. Integrated Treatment of Aqueous Extract of Solanum nigrum-Potentiated Cisplatin- and Doxorubicin-Induced Cytotoxicity in Human Hepatocellular Carcinoma Cells

    PubMed Central

    Wang, Chien-Kai; Lin, Yi-Feng; Tai, Cheng-Jeng; Wang, Chia-Wowi; Chang, Yu-Jia; Choong, Chen-Yen; Lin, Chi-Shian; Tai, Chen-Jei; Chang, Chun-Chao

    2015-01-01

    Chemotherapy is the main approach for treating advanced and recurrent hepatocellular carcinoma (HCC), but the clinical performance of chemotherapy is limited by a relatively low response rate, drug resistance, and adverse effects that severely affect the quality of life of patients. The aqueous extract of Solanum nigrum (AE-SN) is a crucial ingredient in some traditional Chinese medicine (TCM) formulas for treating cancer patients and exhibits antitumor effects in human HCC cells. Therefore, this study examined the tumor-suppression efficiency of AE-SN integrated with a standard chemotherapeutic drug, namely, cisplatin or doxorubicin, in human HCC cells, namely, Hep3B and HepJ5. The results suggested that the integrated treatment with AE-SN-potentiated cisplatin and doxorubicin induced cytotoxicity through the cleavage of caspase-7 and accumulation of microtubule-associated protein-1 light chain-3 A/1B II (LC-3 A/B II), which were associated with apoptotic and autophagic cell death, respectively, in both the Hep3B and HepJ5 cells. In conclusion, AE-SN can potentially be used in novel integrated chemotherapy with cisplatin or doxorubicin to treat HCC patients. PMID:26221175

  12. Cell-specific expression of artificial microRNAs targeting essential genes exhibit potent antitumor effect on hepatocellular carcinoma cells

    PubMed Central

    Mao, Chenyu; Liu, Hao; Chen, Ping; Ye, Jingjia; Teng, Lisong; Jia, Zhenyu; Cao, Jiang

    2015-01-01

    To achieve specific and potent antitumor effect of hepatocyte carcinoma cells, replication defective adenoviral vectors, namely rAd/AFP-amiRG, rAd/AFP-amiRE and rAd/AFP-amiRP, were constructed which were armed with artificial microRNAs (amiRs) targeting essential functional genes glyceraldehyde-3-phosphate dehydrogenase, eukaryotic translation initiation factor 4E and DNA polymerase α respectively under the control of a recombinant promoter comprised of human α-fetoprotein enhancer and basal promoter. The AFP enhancer/promoter showed specific high transcription activity in AFP-positive HCC cells Hep3B, HepG2 and SMMC7721, while low in AFP-negative cell Bcap37. All artificial microRNAs exhibited efficient knockdown of target genes. Decreased ATP production and protein synthesis was observed in rAd/AFP-amiRG and rAd/AFP-amiRE treated HCC cells. All three recombinant adenoviruses showed efficient blockage of cell cycle progression and significant suppression of HCC cells in vitro. In nude mice model bearing Hep3B xenograft, administration of rAd/AFP-amiRG showed potent antitumor effect. The strategy of tumor-specific knockdown of genes essential for cell survival and proliferation may suggest a novel promising approach for HCC gene therapy. PMID:25691059

  13. A Guide to Promoting Resilience in Children: Strengthening the Human Spirit = Guia de Promocion de la Resiliencia en los Ninos para Fortalecer el Espiritu Humano.

    ERIC Educational Resources Information Center

    Grotberg, Edith H.

    Resilience is an important trait because it is the human capacity to face, overcome, be strengthened by, and even transformed by the adversities of life. This guide provides ways to promote resilience in children and help them learn to improve many aspects of their own resilience. The guide is centered on three features, or sources, of resilience:…

  14. CHOQUES AGREGADOS E INVERSIÓN EN CAPITAL HUMANO: EL LOGRO EDUCATIVO SUPERIOR DURANTE LA DÉCADA PERDIDA EN MÉXICO

    PubMed Central

    Peña, Pablo A.

    2014-01-01

    Este artículo documenta una respuesta agregada negativa del logro educativo superior (más de 12 años de escolaridad) en México a la recesión de 1982–83 y el estancamiento que le siguió. La respuesta no fue homogénea entre géneros, regiones y entornos familiares. Los hombres experimentaron una caída en el logro mientras que las mujeres experimentaron un crecimiento más lento. En promedio, los estados con un mayor logro antes del choque experimentaron mayores caídas. La respuesta entre distintos entornos familiares no presenta un patrón claro. Sin embargo, el efecto negativo en el logro se observa incluso entre hermanos. La evidencia sugiere una historia por el lado de la demanda: la caída en el ingreso de los hogares parece ser el determinante de la caída/desaceleración del logro educativo superior. La conclusión es que la recesión y la falta de crecimiento que le siguió tuvieron un efecto negativo importante y duradero en la formación de capacidades en México. PMID:25328251

  15. Estructura y Funcionamiento del Cuerpo Humano. Prontuario. Guia del Maestro. Documento de Trabajo (Structure and Function of the Human Body. Handbook and Teacher's Guide. Working Document).

    ERIC Educational Resources Information Center

    Puerto Rico State Dept. of Education, Hato Rey. Area for Vocational and Technical Education.

    This handbook and teacher's guide are for a 37-week course on the human body, intended for secondary or postsecondary students in allied health occupations. The syllabus lists general objectives and the number of hours and weeks devoted to each unit. A course outline is provided for five units: anatomy and physiology terminology; general…

  16. Serum Dickkopf-1 as a Biomarker for the Diagnosis of Hepatocellular Carcinoma

    PubMed Central

    Kim, Seung Up; Kim, Hyon-Suk; Lee, Jae Myun; Lee, Hyun Gyu; Kim, Hyemi; Choi, Sung Hoon; Baek, Shinhwa; Kim, Beom Kyung; Park, Jun Yong; Kim, Do Young; Ahn, Sang Hoon; Lee, Jong Doo

    2015-01-01

    Purpose Dickkopf-1 (DKK-1) is a Wnt/β-catenin signaling pathway inhibitor. We investigated whether DKK-1 is related to progression in hepatocellular carcinoma (HCC) cells and HCC patients. Materials and Methods In vitro reverse-transcription polymerase chain reaction (RT-PCR), wound healing assays, invasion assays, and ELISAs of patient serum samples were employed. The diagnostic accuracy of the serum DKK-1 ELISA was assessed using receiver operating characteristic (ROC) curves and area under ROC (AUC) analyses. Results RT-PCR showed high DKK-1 expression in Hep3B and low in 293 cells. Similarly, the secreted DKK-1 concentration in the culture media was high in Hep3B and low in 293 cells. Wound healing and invasion assays using 293, Huh7, and Hep3B cells showed that DKK-1 overexpression promoted cell migration and invasion, whereas DKK-1 knock-down inhibited them. When serum DKK-1 levels were assessed in 370 participants (217 with HCC and 153 without), it was significantly higher in HCC patients than in control groups (median 1.48 ng/mL vs. 0.90 ng/mL, p<0.001). The optimum DKK-1 cutoff level was 1.01 ng/mL (AUC=0.829; sensitivity 90.7%; specificity 62.0%). Although DKK-1 had a higher AUC than alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) (AUC=0.829 vs. 0.794 and 0.815, respectively), they were statistically similar (all p>0.05). When three biomarkers were combined (DKK-1 plus AFP plus DCP), they showed significantly higher AUC (AUC=0.952) than single marker, DKK-1 plus AFP, or DKK-1 plus DCP (all p<0.001). Conclusion DKK-1 might be a key regulator in HCC progression and a potential therapeutic target in HCC. Serum DKK-1 could complement the diagnostic accuracy of AFP and DCP. PMID:26256972

  17. Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like particles.

    PubMed

    Ashley, Carlee E; Carnes, Eric C; Phillips, Genevieve K; Durfee, Paul N; Buley, Mekensey D; Lino, Christopher A; Padilla, David P; Phillips, Brandy; Carter, Mark B; Willman, Cheryl L; Brinker, C Jeffrey; Caldeira, Jerri do Carmo; Chackerian, Bryce; Wharton, Walker; Peabody, David S

    2011-07-26

    Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems. PMID:21615170

  18. Cell-Specific Delivery of Diverse Cargos by Bacteriophage MS2 Virus-Like Particles

    PubMed Central

    Ashley, Carlee E.; Carnes, Eric C.; Phillips, Genevieve K.; Durfee, Paul N.; Buley, Mekensey D.; Lino, Christopher A.; Padilla, David P.; Phillips, Brandy; Carter, Mark B.; Willman, Cheryl. L.; Brinker, C. Jeffrey; Caldeira, Jerri do Carmo; Chackerian, Bryce; Wharton, Walker; Peabody, David S.

    2011-01-01

    Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 104-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations < 1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations < 150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to co-display the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill nearly 100% of Hep3B cells (1 × 106 cells/mL population) at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, due to their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems. PMID:21615170

  19. Cell type and context-specific function of PLAG1 for IGF2 P3 promoter activity.

    PubMed

    Akhtar, Monira; Holmgren, Claes; Göndör, Anita; Vesterlund, Mattias; Kanduri, Chandrasekhar; Larsson, Catharina; Ekström, Tomas J

    2012-12-01

    The fetal transcription factor PLAG1 is found to be overexpressed in cancers, and has been suggested to bind the insulin like growth factor 2 (IGF2) P3 promoter, and to activate the IGF2 gene. The expression of IGF2 has partly been linked to loss of CTCF-dependent chromatin insulator function at the H19 imprinting control region (ICR). We investigated the role of PLAG1 for IGF2 regulation in Hep3B and JEG-3 cell lines. Chromatin immunoprecipitation revealed cell type-specific binding of PLAG1 to the IGF2 P3 promoter, which was substantially insensitive to recombinant PLAG1 overexpression in the endogenous context. We hypothesized that the H19 chromatin insulator may be involved in the cell type-specific PLAG1 response. By using a GFP reporter gene/insulator assay plasmid construct with and without the H19 ICR and/or an SV40 enhancer, we confirm that the effect of the insulator is specifically associated with the activity of the IGF2 P3 promoter in the GFP reporter system, and furthermore, that the reporter insulator is functional in JEG-3 but not in Hep3B cells. FACS analysis was used to assess the function of PLAG1 in low endogenously expressing, but Zn-inducible stable PLAG1 expressing JEG-3 cell clones. Considerable increase in IGF2 expression upon PLAG1 induction with a partial insulator overriding activity was found using the reporter constructs. This is in contrast to the effect of the endogenous IGF2 gene which was insensitive to PLAG1 expression in JEG-3, while modestly induced the already highly expressed IGF2 gene in Hep3B cells. We suggest that the PLAG1 binding to the IGF2 P3 promoter and IGF2 expression is cell type-specific, and that the PLAG1 transcription factor acts as a transcriptional facilitator that partially overrides the insulation by the H19 ICR. PMID:23023303

  20. Acasiane A and B and farnesirane A and B, diterpene derivatives from the roots of Acacia farnesiana.

    PubMed

    Lin, An-Shen; Lin, Chia-Rong; Du, Ying-Chi; Lübken, Tilo; Chiang, Michael Y; Chen, I-Hsiao; Wu, Chin-Chung; Hwang, Tsong-Long; Chen, Shu-Li; Yen, Ming-Hong; Chang, Fang-Rong; Wu, Yang-Chang

    2009-02-01

    Four new diterpenes, acasiane A ( 1), acasiane B ( 2), farnesirane A ( 3), and farnesirane B ( 4), along with three known diterpenes ( 5 - 7), two triterpenes ( 8 and 9), and eight flavonoids ( 10 - 17) were isolated from the roots of Acacia farnesiana. The structures and relative configurations of these compounds were determined by various spectroscopic and x-ray analyses. All isolated compounds were evaluated for their in vitro cytotoxic activities against six human cancer cell lines (Hep G2, Hep 3B, MDA-MB-231, MCF-7, A549, and Ca9 - 22) with the MTT method. Betulinic acid ( 8) displayed moderate cytotoxicity (1.70 - 5.74 microg/mL) towards five human cancer cell lines and the flavonoids had slight effects. In addition, 8, diosmetin ( 13), and 3',4',5-trihydroxy-7-methoxyflavone ( 15) slightly inhibited superoxide anion generation or elastase release by human neutrophils, indicating moderate anti-inflammatory activities. PMID:19101886

  1. A new cytotoxic biflavonoid from the rhizome of Wikstroemia indica.

    PubMed

    Shao, Meng; Huang, Xiao-Jun; Liu, Jun-Shan; Han, Wei-Li; Cai, Hong-Bing; Tang, Qing-Fa; Fan, Qin

    2016-06-01

    One new chalcone-flavone biflavonoid, 3'-hydroxydaphnodorin A (1), together with 12 known biflavonoids (2-13), was isolated from the rhizome of Wikstroemia indica. Their structures were established on the basis of extensive spectroscopic methods. Eight isolated compounds 1-3, 6, 7, 9, 12 and 13 were evaluated for their cytotoxic activities against cancer-derived cell lines Hep3B, HepG2 and CNE2, and 1 was found to possess moderate cytotoxicity against HepG2 and CNE2 cell lines, with IC50 values of 65.5 ± 11.4 and 53.6 ± 10.1 μM, respectively. PMID:26252201

  2. Perylenetetracarboxylic anhydride as a precursor of fluorescent carbon nanoonion rings.

    PubMed

    Baldoví, Herme G; Herance, José Raul; Manuel Víctor, Víctor; Alvaro, Mercedes; Garcia, Hermenegildo

    2015-08-01

    Thermal annealing at 400 °C of perylenetetracarboxylic anhydride in low molecular mass PEG gives rise to the formation of well defined nanoobjects of 2.5 nm height and size distribution from 10 to 65 nm (average 40 nm) after purification of the raw mixture with silicagel chromatography. TEM reveals that the flat nanoobjects are constituted of concentric graphenic rings (0.34 nm interlayer distance). The morphology of the nanoparticles resembles onion rings of nanometric dimensions (nanoonion rings C-NOR). C-NOR particles have an excitation dependent emission with λem from 430 to 570 nm and a maximum emission quantum yield of 0.49. C-NOR particles can be internalized into Hep3B human hepatoma cells as determined by confocal fluorescence microscopy and are remarkably biocompatible affecting slightly cell viability according to the MTT test. PMID:26135910

  3. Synthesis and Biological Evaluation of 1-Arylsulfonyl-5-(N-hydroxyacrylamide)indoles as Potent Histone Deacetylase Inhibitors with Antitumor Activity in Vivo

    PubMed Central

    Lai, Mei-Jung; Huang, Han-Li; Pan, Shiow-Lin; Liu, Yi-Min; Peng, Chieh-Yu; Lee, Hsueh-Yun; Yeh, Teng-Kuang; Huang, Po-Hsien; Teng, Che-Ming; Chen, Ching-Shih; Chuang, Hsun-Yueh; Liou, Jing-Ping

    2014-01-01

    A series of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles has been identified as a new class of histone deacetylase inhibitors. Compounds 8, 11, 12, 13, and 14 demonstrated stronger antiproliferative activities than 1 (SAHA) with GI50 values ranging from 0.36 to 1.21 μM against Hep3B, MDA-MB-231, PC-3, and A549 human cancer cell lines. Lead compound 8 showed remarkable HDAC 1, 2, and 6 isoenzymes inhibitory activities with IC50 values of 12.3, 4.0, 1.0 nM, respectively, which are comparable to 1. In in vivo efficacy evaluation against lung A549 xenograft model, 8 displayed better antitumor activity than compound 1. PMID:22439863

  4. Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene.

    PubMed Central

    Pugh, C W; Tan, C C; Jones, R W; Ratcliffe, P J

    1991-01-01

    Erythropoietin, the major hormone controlling red-cell production, is regulated in part through oxygen-dependent changes in the rate of transcription of its gene. Using transient transfection in HepG2 cells, we have defined a DNA sequence, located 120 base pairs 3' to the poly(A)-addition site of the mouse erythropoietin gene, that confers oxygen-regulated expression on a variety of heterologous promoters. The sequence has the typical features of a eukaryotic enhancer. Approximately 70 base pairs are necessary for full activity, but reiteration restores activity to shorter inactive sequences. This enhancer operates in HepG2 and Hep3B cells, but not in Chinese hamster ovary cells or mouse erythroleukemia cells, and responds to cobalt but not to cyanide or 2-deoxyglucose, thus reflecting the physiological control of erythropoietin production accurately. Images PMID:1961720

  5. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation.

    PubMed

    Dzamukova, Maria R; Naumenko, Ekaterina A; Lvov, Yuri M; Fakhrullin, Rawil F

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  6. Merocyanine 540 and Photofrin II as photosensitizers for in vitro killing of duck hepatitis B virus and human hepatoma cells

    NASA Astrophysics Data System (ADS)

    Lin, Tsung-I.; Shien, Yong-Shau; Kao, Ming-Chien

    1994-03-01

    The feasibility of using merocyanine 540 (MC 540) and Photofrin II (PII) as effective photodynamic therapeutic (PDT) agents for killing hepatoma cells and duck hepatitis B virus (DHBV) in vitro was investigated. Cultured duck hepatocytes infected with DHBV and hepatoma cells, Hep 3B and HCC 36, were used as models. MC 540 and PII effectively inhibits the DHBV growth by 90 - 99% in a dose- and light-dependent manner. Photodynamic killing of MC 540 in the two hepatoma cell lines results in 94 - 99% growth inhibition. However, both photosensitizers exhibit dark cytotoxicity (37 - 56%). The present results suggest that MC 540 and PII could be promising and effective photodynamic agents for killing HBV and hepatoma cells.

  7. Cucurbitacin-E inhibits multiple cancer cells proliferation through attenuation of Wnt/β-catenin signaling.

    PubMed

    Feng, Hui; Zang, Li; Zhao, Zhen-Xia; Kan, Quan-Cheng

    2014-06-01

    Recent studies suggest that the use of cucurbitacins could inhibit cancer cell progression. In the current study, the authors analyzed the effect of cucurbitacin-E (CuE) in cancer cells using A549, Hep3B, and SW480 cells. The authors found that CuE inhibited cell proliferation and modulated the expression of cell cycle regulators in these cells. Moreover, the authors found that CuE inhibited Wnt/β-catenin signaling activation through upregulation of tumor suppressor Menin. Indeed, ablation of Menin using small interfering RNA (siRNA) oligos attenuated the antiproliferative roles of CuE. Taken together, the results of this study provide a novel mechanism that may contribute to the antineoplastic effects of CuE in cancer cells. PMID:24885795

  8. Cytotoxic 19-oxygenated steroids from the South China Sea gorgonian, Pacifigorgia senta.

    PubMed

    Chen, Min; Han, Lei; Zhang, Xiu-Li; Wang, Chang-Yun

    2016-06-01

    Bioassay guided chemical investigation of the gorgonian Pacifigorgia senta led to the discovery of a new 19-oxygenated steroid, cholesta-5,24-diene-3β,7β,19-triol (1), as well as three known steroids (2-4). The structure of 1 was determined by extensive spectroscopic analysis, including NMR and MS spectra. All of the compounds exhibited cytotoxicities against HepG2, Hep3B, MCF-7/ADR, PC-3 and HCT-116 cell lines, with the IC50 values ranging from 7.0 to 29.7 μM. It is the first report on the chemical constituents of the coral species P. senta. PMID:26230370

  9. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    PubMed Central

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  10. Ixorapeptide I and ixorapeptide II, bioactive peptides isolated from Ixora coccinea.

    PubMed

    Lee, Chia-Lin; Liao, Yung-Chih; Hwang, Tsong-Long; Wu, Chin-Chung; Chang, Fang-Rong; Wu, Yang-Chang

    2010-12-15

    Two novel derivatized peptides, designated as ixorapeptide I (1) and ixorapeptide II (2), in addition to 28 other known compounds, were isolated from the MeOH extract of Ixora coccinea using bioassay-guided fractionation. The structures of metabolites 1 and 2 were determined by interpretation of the spectroscopic data and Marfey's method. Compound 1 exhibited selective potency against Hep3B liver cancer cell line with an IC(50) value of 3.36 μg/mL, and compound 2 did not show notable cytotoxicity toward cancer cell lines but could inhibit superoxide anion generation and elastase release with IC(50) values of 0.21 and 0.27 μg/mL, respectively. Moreover, kaempferol and luteolin from this plant showed inhibition with IC(50) values of 3.55 and 2.56 μg/mL, respectively on platelet aggregation induced by collagen. PMID:21106454

  11. Perylenetetracarboxylic anhydride as a precursor of fluorescent carbon nanoonion rings

    NASA Astrophysics Data System (ADS)

    Baldoví, Herme G.; Herance, José Raul; Manuel Víctor, Víctor; Alvaro, Mercedes; Garcia, Hermenegildo

    2015-07-01

    Thermal annealing at 400 °C of perylenetetracarboxylic anhydride in low molecular mass PEG gives rise to the formation of well defined nanoobjects of 2.5 nm height and size distribution from 10 to 65 nm (average 40 nm) after purification of the raw mixture with silicagel chromatography. TEM reveals that the flat nanoobjects are constituted of concentric graphenic rings (0.34 nm interlayer distance). The morphology of the nanoparticles resembles onion rings of nanometric dimensions (nanoonion rings C-NOR). C-NOR particles have an excitation dependent emission with λem from 430 to 570 nm and a maximum emission quantum yield of 0.49. C-NOR particles can be internalized into Hep3B human hepatoma cells as determined by confocal fluorescence microscopy and are remarkably biocompatible affecting slightly cell viability according to the MTT test.

  12. N-Sulfonyl-aminobiaryls as Antitubulin Agents and Inhibitors of Signal Transducers and Activators of Transcription 3 (STAT3) Signaling.

    PubMed

    Lai, Mei-Jung; Lee, Hsueh-Yun; Chuang, Hsun-Yueh; Chang, Li-Hsun; Tsai, An-Chi; Chen, Mei-Chuan; Huang, Han-Lin; Wu, Yi-Wen; Teng, Che-Ming; Pan, Shiow-Lin; Liu, Yi-Min; Mehndiratta, Samir; Liou, Jing-Ping

    2015-08-27

    A series of N-sulfonyl-aminobiaryl derivatives have been examined as novel antitubulin agents. Compound 21 [N-(4'-cyano-3'-fluoro-biphenyl-2-yl)-4-methoxy-benzenesulfonamide] exhibits remarkable antiproliferative activity against four cancer cell lines (pancreatic AsPC-1, lung A549, liver Hep3B, and prostate PC-3) with a mean GI50 value of 57.5 nM. Additional assays reveal that 21 inhibits not only tubulin polymerization but also the phosphorylation of STAT3 inhibition with an IC50 value of 0.2 μM. Four additional compounds (8, 10, 19, and 35) are also able to inhibit this phosphorylation. This study describes novel N-sulfonyl-aminobiaryl (biaryl-benzenesulfonamides) as potent anticancer agents targeting both STAT3 and tubulin. PMID:26241032

  13. Inhibitory efficacy of the quantified prunellae spica extract on H22 tumor bearing mice

    NASA Astrophysics Data System (ADS)

    Wang, Zhi-ping; Chen, Tong-sheng

    2013-02-01

    Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistence of the advanced hepatocarcinoma to chemotherapy. In this report, we assessed the antitumor activity of a prunellae spica aqueous extract (PSE) in vitro and in vivo. PSE was quantified by HPLC and UV. MTT assay showed that PSE did not effectively inhibit the growth of H22 cells. The in vivo anti-tumor activity was assessed by using the mice bearing H22 tumor. In vivo studies showed the higher antitumor efficacy of PSE without significant side effect assessed by the reduced tumor weight, and the extended survival time of the mice bearing H22 solid and ascites tumor. Collectively, PSE is a promising Chinese medicinal herb for treating hepatocarcinoma.

  14. A base substitution in the promoter associated with the human haptoglobin 2-1 modified phenotype decreases transcriptional activity and responsiveness to interleukin-6 in human hepatoma cells

    SciTech Connect

    Grant, D.J.; Maeda, N. )

    1993-05-01

    An A-to-C base substitution at nucleotide position -61 in the promoter region of the human haptoglobin gene (Hp) has been shown to be strongly associated with the haptoglobin 2-1 modified (Hp2-1mod) phenotype. In order to investigate whether this base substitution is the cause of reduced expression of the Hp[sup 2] allele relative to the Hp[sup 1] allele in individuals with the Hp2-1mod phenotype, the authors used the chloramphenicol acetyl transferase (CAT) expression system to evaluate promoter function. In HepG2 cells, which normally express their endogenous haptoglobin genes, CAT plasmid constructs with the -61C base change in the promoter had about 10-fold-lower transcriptional activity after transfection than did the Hp control construct. The -61C substitution also rendered the construct unresponsive to treatment by interleukin-6 after transfection into Hep3B2 cells, which normally do not express haptoglobin but do so in response to stimulation by acute-phase reactants. In addition, two base substitutions, T to A and A to G, at positions -104 and -55G, respectively, in the promoter region of the Hp[sup 1] allele, are also associated with the Hp2-1mod phenotype. CAT constructs with both substitutions (-104A-55G) and with one substitution (-55G) showed activity similar to that in the Hp control when transfected into both HepG2 and Hep3B2 cells, although interleukin-6 induction was less than with the Hp control construct. These results further support the hypothesis that the Hp2-1mod phenotype results, in part, from the -61C mutation in the promoter region of the Hp[sup 2] gene.

  15. Caspase mediated synergistic effect of Boswellia serrata extract in combination with doxorubicin against human hepatocellular carcinoma.

    PubMed

    Khan, Mohammad Ahmed; Singh, Mhaveer; Khan, Masood Shah; Najmi, Abul Kalam; Ahmad, Sayeed

    2014-01-01

    The study investigated the growth-inhibiting and apoptosis mediating effects of B. serrata extract as monotherapy and combination therapy with DOX against hepatocellular carcinoma cell lines. Boswellic acid rich fraction of B. serrata extract was prepared. MTT assay on HepG2 and Hep3B cells was carried out using B. serrata alone and in combination with DOX. Further, caspase-3 activity, TNF-α level, and IL-6 level were estimated. Isobolographic analysis was carried out to evaluate the effect of combination therapy. Additionally, protective effect of B. serrata extract on DOX induced hepatic toxicity was also evaluated in Wistar rats. B. serrata extract inhibited growth of HepG2 (IC50 value of 21.21 ± 0.92 μg/mL) as well as HepG2 (IC50 value of 18.65 ± 0.71 μg/mL). DOX inhibited growth in HepG2 and Hep3B cells with an IC50 of 1.06 ± 0.04 μg/mL and 1.92 ± 0.09 μg/mL. Isobolographic analysis showed combination index (CI) of DOX and B. serrata extract of 0.53 ± 0.03 to 0.79 ± 0.02 suggesting synergistic behavior against the two cell lines. B. serrata extract also caused dose dependent increase in caspase-3 activity, TNF-α level, and IL-6 level which was higher (P < 0.001) with DOX (1 μM) and B. serrata extract (20 μg/mL) combination. B. serrata extract also protected Wistar rats against DOX induced hepatic toxicity. PMID:25177685

  16. Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma.

    PubMed

    Djojosubroto, Meta W; Chin, Allison C; Go, Ning; Schaetzlein, Sonja; Manns, Michael P; Gryaznov, Sergei; Harley, Calvin B; Rudolph, K Lenhard

    2005-11-01

    Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy. PMID:16114043

  17. The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

    PubMed

    Gahr, Susanne; Mayr, Christian; Kiesslich, Tobias; Illig, Romana; Neureiter, Daniel; Alinger, Beate; Ganslmayer, Marion; Wissniowski, Till; Fazio, Pietro Di; Montalbano, Roberta; Ficker, Joachim H; Ocker, Matthias; Quint, Karl

    2015-09-01

    Post-translational modifications of chromatin components are significantly involved in the regulation of tumor suppressor gene and oncogene expression. Connective tissue growth factor (CTGF) is an epigenetically regulated growth factor with functions in angiogenesis and cell-matrix interactions and plays a pivotal role in hepatocellular carcinoma (HCC). The pharmacologic inhibition of histone and protein deacetylases represents a new approach to interfere with pathways of apoptosis and angiogenesis. We investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on human HCC cell lines HepG2 (p53wt) and Hep3B (p53null) and in a subcutaneous xenograft model and explored the influence on angiogenesis. Specimens were characterized by quantitative real-time PCR. Protein was separated for western blotting against CTGF, VEGF, VEGF receptor-1 (VEGFR-1/FLT-1), VEGF receptor-2 (VEGFR-2/KDR), MAPK and phospho-MAPK. In vivo, HepG2 cells were xenografted to NMRI mice and treated with daily i.p. injections of 10 mg/kg panobinostat. After 1, 7 and 28 days, real-time PCR was performed. Immunohistochemistry and western blotting were examined after 28 days. An increased significant expression of CTGF was only seen after 24 h treatment with 0.1 µM panobinostat in HepG2 cells and Hep3B cells, whereas after 72 h treatment CTGF expression clearly decreased. In the xenografts, treatment with panobinostat showed a minimal CTGF expression after 1 day and 4 weeks, respectively. In vitro as well as in vivo, VEGF was not affected by panobinostat treatment at any time. In conclusion, panobinostat influences extracellular signaling cascades via CTGF-dependent pathways. PMID:26202945

  18. New Approach for Treatment of Primary Liver Tumors: The Role of Quercetin.

    PubMed

    Brito, Ana Filipa; Ribeiro, Marina; Abrantes, Ana Margarida; Mamede, Ana Catarina; Laranjo, Mafalda; Casalta-Lopes, João Eduardo; Gonçalves, Ana Cristina; Sarmento-Ribeiro, Ana Bela; Tralhão, José Guilherme; Botelho, Maria Filomena

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumor (PLT), with cholangiocarcinoma (CC) being the second most frequent. Glucose transporter 1 (GLUT-1) expression is increased in PLTs and therefore it is suggested as a therapeutic target. Flavonoids, like quercetin, are GLUT-1 competitive inhibitors and may be considered as potential therapeutic agents for PLTs. The objective of this study was evaluation of quercetin anticancer activity in three human HCC cell lines (HepG2, HuH7, and Hep3B2.1-7) and in a human CC cell line (TFK-1). The possible synergistic effect between quercetin and sorafenib, a nonspecific multikinase inhibitor used in clinical practice in patients with advanced HCC, was also evaluated. It was found that in all the cell lines, quercetin induced inhibition of the metabolic activity and cell death by apoptosis, followed by increase in BAX/BCL-2 ratio. Treatment with quercetin caused DNA damage in HepG2, Hep3B2.1-7, and TFK-1 cell lines. The effect of quercetin appears to be independent of P53. Incubation with quercetin induced an increase in GLUT-1 membrane expression and a consequent reduction in the cytoplasmic fraction, observed as a decrease in (18)F-FDG uptake, indicating a GLUT-1 competitive inhibition. The occurrence of synergy when sorafenib and quercetin were added simultaneously to HCC cell lines was noticed. Thus, the use of quercetin seems to be a promising approach for PLTs through GLUT-1 competitive inhibition. PMID:26943884

  19. HCA519/TPX2: a potential T-cell tumor-associated antigen for human hepatocellular carcinoma

    PubMed Central

    Aref, Ahmed M; Hoa, Neil T; Ge, Lisheng; Agrawal, Anshu; Dacosta-Iyer, Maria; Lambrecht, Nils; Ouyang, Yi; Cornforth, Andrew N; Jadus, Martin R

    2014-01-01

    Background Immunotherapy for human hepatocellular cancer (HCC) is slowly making progress towards treating these fatal cancers. The identification of new antigens can improve this approach. We describe a possible new antigen, hepatocellular carcinoma‐associated antigen‐519/targeting protein for Xklp‐2 (HCA519/TPX2), for HCC that might be beneficial for T‐cell specific HCC immunotherapy. Methods HCC was studied for the expression for 15 tumor‐associated antigens considered useful for immunotherapy within three HCC cell lines (HepG2, Hep3B, and PLC/PRF/5), lymphocytes, non‐cancerous livers, and clinical HCC. The expression of tumor antigenic precursor proteins (TAPPs) messenger RNA was first screened by reverse transcriptase quantitative real‐time polymerase chain reaction. Results Four antigens (alpha fetoprotein, aspartyl/asparaginyl β­hydroxylase, glypican­3 and HCA519/TPX2) proved to be the best expressed TAPPs within the HCC specimens by molecular analyses. HCA519/TPX2 was detected by intracellular cell flow cytometry within HCC cell lines by using a specific antibody towards this TAPP. This antibody also detected the protein within primary HCCs. We synthesized two HCA519/TPX2 peptides (HCA519464–472 and HCA519351–359) which can bind to human leukocyte antigen (HLA)‐A*0201. Dendritic cells pulsed with these peptides stimulated cytolytic T lymphocytes (CTLs). These killer T‐cells lysed HLA‐A*0201+ T2 cells exogenously loaded with the correct specific peptide. The CTLs killed HepG2 (HLA‐A2+ and HCA519+), but not the Hep3B and PLC/PRF/5 cell lines, which are HCA519+ but HLA‐A2‐negative. In silico analysis reveals that HCA519/TPX2 has the inherent ability to bind to a very wide variety of HLA antigens. Conclusion HCA519/TPX2 is a viable immunotarget that should be further investigated within HCC patients. PMID:24966688

  20. Loss of Runt-related transcription factor 3 induces resistance to 5-fluorouracil and cisplatin in hepatocellular carcinoma.

    PubMed

    Kataoka, Junro; Shiraha, Hidenori; Horiguchi, Shigeru; Sawahara, Hiroaki; Uchida, Daisuke; Nagahara, Teruya; Iwamuro, Masaya; Morimoto, Hiroki; Takeuchi, Yasuto; Kuwaki, Kenji; Onishi, Hideki; Nakamura, Shinichiro; Takaki, Akinobu; Nouso, Kazuhiro; Yagi, Takahito; Yamamoto, Kazuhide; Okada, Hiroyuki

    2016-05-01

    Runt-related transcription factor 3 (RUNX3) is known to function as a tumor suppressor in gastric cancer and other types of cancers, including hepatocellular carcinoma (HCC). However, its role has not been fully elucidated. In the present study, we aimed to evaluate the role of RUNX3 in HCC. We used the human HCC cell lines Hep3B, Huh7 and HLF; RUNX3 cDNA was introduced into Hep3B and Huh7 cells, which were negative for endogenous RUNX3 expression, and RUNX3 siRNA was transfected into HLF cells, which were positive for endogenous RUNX3. We analyzed the expression of RUNX3 and multidrug resistance-associated protein (MRP) by immunoblotting. MTT assays were used to determine the effects of RUNX3 expression on 5-fluorouracil (5-FU) and cisplatin (CDDP) sensitivity. Finally, 23 HCC specimens resected from patients with HCC at Okayama University Hospital were analyzed, and correlations among immunohistochemical expression of RUNX3 protein and MRP protein were evaluated in these specimens. Exogenous RUNX3 expression reduced the expression of MRP1, MRP2, MRP3 and MRP5 in the RUNX3-negative cells, whereas knockdown of RUNX3 in the HLF cells stimulated the expression of these MRPs. An inverse correlation between RUNX3 and MRP expression was observed in the HCC tissues. Importantly, loss of RUNX3 expression contributed to 5-FU and CDDP resistance by inducing MRP expression. These data have important implications in the study of chemotherapy resistance in HCC. PMID:26985715

  1. Synthesis, characterization and in vitro anticancer activity of 18-membered octaazamacrocyclic complexes of Co(II), Ni(II), Cd(II) and Sn(II)

    NASA Astrophysics Data System (ADS)

    Kareem, Abdul; Zafar, Hina; Sherwani, Asif; Mohammad, Owais; Khan, Tahir Ali

    2014-10-01

    An effective series of 18 membered octaazamacrocyclic complexes of the type [MLX2], where X = Cl or NO3 have been synthesized by template condensation reaction of oxalyl dihydrazide with dibenzoylmethane and metal salt in 2:2:1 molar ratio. The formation of macrocyclic framework, stereochemistry and their overall geometry have been characterized by various physico-chemical studies viz., elemental analysis, electron spray ionization-mass spectrometry (ESI-MS), I.R, UV-Vis, 1H NMR, 13C NMR spectroscopy, X-ray diffraction (XRD) and TGA/DTA studies. These studies suggest formation of octahedral macrocyclic complexes of Co(II), Ni(II), Cd(II) and Sn(II). The molar conductance values suggest nonelectrolytic nature for all the complexes. Thermogravimatric analysis shows that all the complexes are stable up to 600 °C. All these complexes have been tested against different human cancer cell lines i.e. human hepatocellular carcinoma (Hep3B), human cervical carcinoma (HeLa), human breast adenocarcinoma (MCF7) and normal cells (PBMC). The newly synthesized 18-membered octaazamacrocyclic complexes during in vitro anticancer evaluation, displayed moderate to good cytotoxicity on liver (Hep3B), cervical (HeLa) and breast (MCF7) cancer cell lines, respectively. The most effective anticancer cadmium complex (C34H28N10CdO10) was found to be active with IC50 values, 2.44 ± 1.500, 3.55 ± 1.600 and 4.82 ± 1.400 in micro-molar on liver, cervical and breast cancer cell lines, respectively.

  2. A 24-base-pair sequence 3' to the human erythropoietin gene contains a hypoxia-responsive transcriptional enhancer.

    PubMed Central

    Madan, A; Curtin, P T

    1993-01-01

    Erythropoietin (Epo) synthesis increases in response to hypoxia. The hepatoma cell line Hep 3B produces low basal levels of Epo mRNA which increase markedly with hypoxia. To define the sequences necessary for this response, we linked fragments of the human Epo gene to a luciferase vector, introduced these plasmids into Hep 3B cells and assayed for luciferase activity after growth in 1% or 21% oxygen. A 621-bp Epo promoter fragment resulted in a 2.4-fold increase in luciferase activity with hypoxia. We tested several Epo gene fragments upstream of this Epo promoter fragment and found that a 613-bp Bgl II-Pvu II 3' fragment had a 10-fold increase in activity with hypoxia regardless of orientation. This fragment had a similar level of activity when linked to a simian virus 40 promoter. Portions of this fragment retained activity, including a 38-bp Apa I-Taq I fragment that had a 17-fold increase in activity with hypoxia. Deletion of nt 4-13 or 19-28 from this 38-bp fragment resulted in a loss of activity. The 24-bp upstream portion of the 38-bp fragment showed an 8-fold increase in activity with hypoxia. However, deletion of nt 19-24 or mutagenesis of nt 21 or 22 in this 24-bp fragment resulted in loss of activity. Our studies indicate that the transcriptional response of the human Epo gene to hypoxia is mediated in part by promoter sequences and to a greater degree by an enhancer element located in a 24-bp portion of the 3' flanking sequence of the gene. PMID:8387202

  3. Triple-controlled oncolytic adenovirus expressing melittin to exert inhibitory efficacy on hepatocellular carcinoma

    PubMed Central

    Qian, Chun-Yu; Wang, Kai-Li; Fang, Fan-Fu; Gu, Wei; Huang, Feng; Wang, Fu-Zhe; Li, Bai; Wang, Li-Na

    2015-01-01

    Hepatocellular carcinoma (HCC) is a highly malignant disease, and its outcome of routine therapies is poor. Comprehensive treatment including gene therapy is an important way to improve patients’ prognosis and survival. In this study, we successfully constructed a triple-controlled cancer-selective oncolytic adenovirus, QG511-HA-Melittin, carrying melittin gene, in which the hybrid promoter, hypoxia-response element (HRE)-AFP promoter, was used to control viral E1a expression targeting AFP-positive cancer cells in hypoxia microenviroment, and the E1b-55 kDa gene was deleted in cancer cells with p53-deficiency. The cytological experiments found that the viral replication of QG511-HA-Melittin was increased to 12800-folds in Hep3B cells within 48 h, and 130-folds in SMMC-7721, but the virus did not replicate in L-02 cells. QG511-HA-Melittin had a strong inhibition effect on AFP-positive HCC cell proliferation, such as Hep3B and HepG2, whereas, there was low or no inhibition effect of QG511-HA-Melittin on AFP-negative cancer cells SMMC-7721 and normal cells L-02. In the in vivo experiment, compared with the blank control group, QG511-HA-Melittin can significantly inhibit the growth of HCC xenografts (P<0.05). The survival of mice in QG511-HA-Melittin group was much longer than that of the blank control group. Both in vitro and in vivo experiments manifested that QG511-HA-Melittin exerts an inhibitory effect on HCC cells, which may provide a new strategy for HCC biotherapy. PMID:26617748

  4. Loss of Runt-related transcription factor 3 induces resistance to 5-fluorouracil and cisplatin in hepatocellular carcinoma

    PubMed Central

    KATAOKA, JUNRO; SHIRAHA, HIDENORI; HORIGUCHI, SHIGERU; SAWAHARA, HIROAKI; UCHIDA, DAISUKE; NAGAHARA, TERUYA; IWAMURO, MASAYA; MORIMOTO, HIROKI; TAKEUCHI, YASUTO; KUWAKI, KENJI; ONISHI, HIDEKI; NAKAMURA, SHINICHIRO; TAKAKI, AKINOBU; NOUSO, KAZUHIRO; YAGI, TAKAHITO; YAMAMOTO, KAZUHIDE; OKADA, HIROYUKI

    2016-01-01

    Runt-related transcription factor 3 (RUNX3) is known to function as a tumor suppressor in gastric cancer and other types of cancers, including hepatocellular carcinoma (HCC). However, its role has not been fully elucidated. In the present study, we aimed to evaluate the role of RUNX3 in HCC. We used the human HCC cell lines Hep3B, Huh7 and HLF; RUNX3 cDNA was introduced into Hep3B and Huh7 cells, which were negative for endogenous RUNX3 expression, and RUNX3 siRNA was transfected into HLF cells, which were positive for endogenous RUNX3. We analyzed the expression of RUNX3 and multidrug resistance-associated protein (MRP) by immunoblotting. MTT assays were used to determine the effects of RUNX3 expression on 5-fluorouracil (5-FU) and cisplatin (CDDP) sensitivity. Finally, 23 HCC specimens resected from patients with HCC at Okayama University Hospital were analyzed, and correlations among immunohistochemical expression of RUNX3 protein and MRP protein were evaluated in these specimens. Exogenous RUNX3 expression reduced the expression of MRP1, MRP2, MRP3 and MRP5 in the RUNX3-negative cells, whereas knockdown of RUNX3 in the HLF cells stimulated the expression of these MRPs. An inverse correlation between RUNX3 and MRP expression was observed in the HCC tissues. Importantly, loss of RUNX3 expression contributed to 5-FU and CDDP resistance by inducing MRP expression. These data have important implications in the study of chemotherapy resistance in HCC. PMID:26985715

  5. Targeting of the Akt-Nuclear Factor-κB Signaling Network by [1-(4-Chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a Novel Indole-3-Carbinol Derivative, in a Mouse Model of Hepatocellular Carcinoma

    PubMed Central

    Omar, Hany A.; Sargeant, Aaron M.; Weng, Jing-Ru; Wang, Dasheng; Kulp, Samuel K.; Patel, Tushar

    2009-01-01

    Constitutive activation of Akt and nuclear factor-κB (NF-κB) represents major cellular abnormalities associated with the development and progression of hepatocellular carcinoma (HCC). Based on the structure of indole-3-carbinol, a chemopreventive phytochemical, we developed a novel derivative, [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), that exhibits higher potency in inducing apoptosis by targeting the Akt-NF-κB signaling network. This study was aimed at assessing the antitumor activity of OSU-A9 using both in vitro and in vivo models of HCC, a malignancy in which the Akt-NF-κB signaling network plays major roles in pathogenesis and therapeutic resistance. Our data show that OSU-A9 was 100 times more potent than indole-3-carbinol in suppressing the viability of Hep3B, Huh7, and PLC5 HCC cells with IC50 values ranging from 2.8 to 3.2 μM. OSU-A9 interfered with the interplay between Akt- and NF-κB-mediated oncogenic signaling, leading to changes in the functional status of diverse signaling effectors involved in cell cycle progression, apoptosis, angiogenesis, and metastasis. The in vivo efficacy of OSU-A9 was assessed in nude mice bearing luciferase-expressing Hep3B xenograft tumors. Daily oral treatments with OSU-A9 at 25 or 50 mg/kg for 56 days suppressed tumor growth by 67 and 80%, respectively, which was correlated with changes in intratumoral biomarkers pertinent to Akt-NF-κB signaling, and without apparent toxicity or evidence of hepatic biotransformation enzyme induction. Together, these findings indicate that OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-κB signaling network with a broad spectrum of antitumor activity that includes targets regulating multiple aspects of HCC pathogenesis and progression. PMID:19706731

  6. Suppression of AKT Anti-Apoptotic Signaling by a Novel Drug Candidate Results in Growth Arrest and Apoptosis of Hepatocellular Carcinoma Cells

    PubMed Central

    Cuconati, Andrea; Mills, Courtney; Goddard, Cally; Zhang, Xianchao; Yu, Wenquan; Guo, Haitao; Xu, Xiaodong; Block, Timothy M.

    2013-01-01

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer fatalities worldwide, with limited treatment options and five year survival rates of between <5 and 15%. To address this medical need, we conducted a screen of a drug-like small molecule library for HCC-selective cytotoxins. We report here the identification of a disubstituted aminothiazole termed HBF-0079, with remarkable selective toxicity for HCC-derived cell lines versus non-HCC liver lines and most other cancer lines. HBF-0079 caused irreversible growth arrest and apoptosis of the HCC lines Huh7, Hep3B, HepaRG as well as the hepatoblastoma line HepG2, with CC50 values from ∼0.7−7.7 µM, while more than 45 µM was needed to achieve CC50 values for the immortalized normal hepatocyte lines THLE-2 and PH5CH. Of the sixty cancer lines from the National Cancer Institute panel, only five exhibited >50% growth inhibition by HBF-0079. In Huh7 cells, HBF-0079 induced cell cycle arrest in G1 and concomitant apoptosis, and its effects were irreversible after removal of the compound. These observations corroborate a loss of AKT phosphorylation at the mTORC2-targeted residue S473, with concurrent loss of phosphorylation of the mTORC1 targets SK6 and 4EBP1 in Huh7 but not PH5CH cells. Finally, growth of Hep3B-derived tumors in a murine xenograft model was significantly repressed by the compound through either systemic or intratumoral administration of formulated HBF-0079. The potential for development of this drug candidate is discussed. PMID:23355882

  7. CF102 an A3 Adenosine Receptor Agonist Mediates Anti-Tumor and Anti-Inflammatory Effects in the Liver

    PubMed Central

    COHEN, S.; STEMMER, S.M.; ZOZULYA, G.; OCHAION, A.; PATOKA, R.; BARER, F.; BAR-YEHUDA, S.; RATH-WOLFSON, L.; JACOBSON, K.A.; FISHMAN, P.

    2012-01-01

    The Gi protein-associated A3 adenosine receptor (A3AR) is a member of the adenosine receptor family. Selective agonists at the A3AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 μg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions. PMID:21660967

  8. CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver.

    PubMed

    Cohen, S; Stemmer, S M; Zozulya, G; Ochaion, A; Patoka, R; Barer, F; Bar-Yehuda, S; Rath-Wolfson, L; Jacobson, K A; Fishman, P

    2011-09-01

    The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions. PMID:21660967

  9. Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2010-12-01

    Gradients of Po(2) between capillary blood and mitochondria are the driving force for diffusional O(2) delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O(2) gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O(2) gradients, we developed a novel technique that permits imaging of intracellular O(2) levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O(2) ≤3%, by the O(2)-dependent red shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a two-dimensional O(2) diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O(2) diffusion from the top surface of the cell layer. The magnitude of the red shift progressively increased as the distance from the gas coverslip interface increased. It reached an anoxic level in cells located at ∼220 μm and ∼690 μm from the gas coverslip boundary at 1% and 3% gas phase O(2), respectively. Thus the average O(2) gradient was 0.03 mmHg/μm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red shift in mitochondria-targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O(2) gradients is intracellular. Findings in the present two-dimensional O(2) diffusion model support the crucial role of tissue O(2) diffusion in defining the O(2) microenvironment in individual cells. PMID:20844249

  10. SIN3A, Generally Regarded as a Transcriptional Repressor, Is Required for Induction of Gene Transcription by the Aryl Hydrocarbon Receptor*

    PubMed Central

    Solaimani, Parrisa; Wang, Feng; Hankinson, Oliver

    2014-01-01

    CYP1A1 bioactivates several procarcinogens and detoxifies several xenobiotic compounds. Transcription of CYP1A1 is highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor. We recently described an RNAi high throughput screening performed in the Hepa-1 mouse hepatoma cell line, which revealed that SIN3A is necessary for the induction of CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) enzymatic activity by TCDD. In the current studies, we sought to provide insight into the role of SIN3A in this process, particularly because studies on SIN3A have usually focused on its repressive activity on transcription. We report that ectopic expression of human SIN3A in Hepa-1 cells enhanced EROD induction by TCDD and efficiently rescued TCDD induction of EROD activity in cells treated with an siRNA to mouse SIN3A, thus validating a role for SIN3A in CYP1A1 induction. We demonstrate that SIN3A is required for TCDD induction of the CYP1A1 protein in Hepa-1 cells but not for expression of the aryl hydrocarbon receptor protein. In addition, siRNAs for SIN3A decreased TCDD-mediated induction of CYP1A1 mRNA and EROD activity in human hepatoma cell line Hep3B. We establish that TCDD treatment of Hepa-1 cells rapidly increases the degree of SIN3A binding to both the proximal promoter and enhancer of the Cyp1a1 gene and demonstrate that increased binding to the promoter also occurs in human Hep3B, HepG2, and MCF-7 cells. These studies establish that SIN3A physically interacts with the CYP1A1 gene and extends the transcriptional role of SIN3A to a gene that is very rapidly and dramatically induced. PMID:25305016

  11. The role of APE/Ref-1 signaling pathway in hepatocellular carcinoma progression

    PubMed Central

    YANG, ZHEN; YANG, SUN; MISNER, BOBBYE J.; LIU-SMITH, FENG; MEYSKENS, FRANK L.

    2014-01-01

    Hepatocellular carcinoma (HCC) is responsible for a third of the estimated cancer-caused deaths worldwide. To deeply understand the mechanisms controlling HCC progression is of primary importance to develop new approaches for treatment. Apurinic/apyrimidinic endonuclease-1/redox effector factor 1 (APE/Ref-1) has been uncovered elevated in various types of cancer, including HCC. Additionally, HCC progression is always correlated with elevated copper (Cu). Our previous data demonstrated that Cu treatment initiated APE/Ref-1 expression and its downstream targets. Therefore, we hypothesized that APE/Ref-1 may be involved in HCC progression through mediating the effect of Cu to its signaling cascades. Following different treatments, human HCC cell line (Hep3B) and immortalized non-malignant hepatocyte cell line (THLE3) were analyzed to explore the role of APE/Ref-1 signaling pathway. Unstained human tissue microarrays (TMA) were subjected to IHC analysis to study the relationship between APE/Ref-1 expression and clinic features. APE/Ref-1 was upregulated in HCC cells consistent with the strong expression of APE/Ref-1 in HCC tissue microarray. Greater cytoplasmic accumulation of APE/Ref-1 was found in poorly differentiated and more aggressive tumors. Also we provide evidence to show that APE/Ref-1 signaling pathway stimulates cellular proliferation, enhances antiapoptosis, and facilitates metastasis through experimental knockdown of APE/Ref-1 using siRNA in Hep3B cells or overexpressing APE/Ref-1 in THLE3 cells. These results define a novel role of APE/Ref-1 in HCC progression as being an important mediating and potentiating molecule, and also provide a basis for further investigations utilizing appropriate APE/Ref-1 inhibitors in combination with chemo-drugs for HCC treatment. PMID:25109342

  12. Autophagy-related cell death by pan-histone deacetylase inhibition in liver cancer.

    PubMed

    Di Fazio, Pietro; Waldegger, Petra; Jabari, Samir; Lingelbach, Susanne; Montalbano, Roberta; Ocker, Matthias; Slater, Emily P; Bartsch, Detlef K; Illig, Romana; Neureiter, Daniel; Wissniowski, Thaddeus T

    2016-05-17

    Autophagy is a homeostatic, catabolic degradation process and cell fate essential regulatory mechanism. Protracted autophagy triggers cell death; its aberrant function is responsible for several malignancies. Panobinostat, a potent pan-deacetylase inhibitor, causes endoplasmic reticulum stress-induced cell death. The aim of this study was to investigate the role of autophagy in deacetylase inhibitor-triggered liver cancer cell death.HepG2 (p53wt) and Hep3B (p53 null) liver cancer cell lines were exposed to panobinostat. RT-qPCR and western blot confirmed autophagic factor modulation. Immuno-fluorescence, -precipitation and -histochemistry as well as transmission electron microscopy verified autophagosome formation. The cytotoxicity of panobinostat and autophagy modulators was detected using a real time cell viability assay.Panobinostat induced autophagy-related factor expression and aggregation. Map1LC3B and Beclin1 were significantly over-expressed in HepG2 xenografts in nude mice treated with panobinostat for 4 weeks. Subcellular distribution of Beclin1 increased with the appearance of autophagosomes-like aggregates. Cytosolic loss of p53, in HepG2, and p73, in Hep3B cells, and a corresponding gain of their nuclear level, together with modulation of DRAM1, were observed. Autophagosome aggregation was visible after 6 h of treatment. Treatment of cells stably expressing GFP-RFPtag Map1LC3B resulted in aggregation and a fluorescence switch, thus confirming autophagosome formation and maturation. Tamoxifen, an inducer of autophagy, caused only a block in cell proliferation; but in combination with panobinostat it resulted in cell death.Autophagy triggers cell demise in liver cancer. Its modulation by the combination of tamoxifen and panobinostat could be a new option for palliative treatment of hepatocellular carcinoma. PMID:27058414

  13. Effect of Ottoman Viper (Montivipera xanthina (Gray, 1849)) Venom on Various Cancer Cells and on Microorganisms.

    PubMed

    Yalcın, Husniye Tansel; Ozen, Mehmet Ozgün; Gocmen, Bayram; Nalbantsoy, Ayse

    2014-01-01

    Cytotoxic and antimicrobial effects of Montivipera xanthina venom against LNCaP, MCF-7, HT-29, Saos-2, Hep3B, Vero cells and antimicrobial activity against selected bacterial and fungal species: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, E. coli O157H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus epidermidis ATCC 12228, S. typhimirium CCM 5445, Proteus vulgaris ATCC 6957 and Candida albicans ATCC 10239 were studied for evaluating the potential medical benefit of this snake venom. Cytotoxicity of venom was determined using MTT assay. Snake venom cytotoxicity was expressed as the venom dose that killed 50 % of the cells (IC50). The antimicrobial activity of venom was studied by minimal inhibitory concentration (MIC) and disc diffusion assay. MIC was determined using broth dilution method. The estimated IC50 values of venom varied from 3.8 to 12.7 or from 1.9 to 7.2 μg/ml after treatment with crude venom for 24 or 48 h for LNCaP, MCF-7, HT-29 and Saos-2 cells. There was no observable cytotoxic effect on Hep3B and Vero cells. Venom exhibited the most potent activity against C. albicans (MIC, 7.8 μg/ml and minimal fungicidal concentration, 62.5 μg/ml) and S. aureus (MIC, 31.25 μg/ml). This study is the first report showing the potential of M. xanthina venom as an alternative therapeutic approach due to its cytotoxic and antimicrobial effects. PMID:23381026

  14. Interaction of HIF and USF signaling pathways in human genes flanked by hypoxia-response elements and E-box palindromes.

    PubMed

    Hu, Junmin; Stiehl, Daniel P; Setzer, Claudia; Wichmann, Daniela; Shinde, Dheeraj A; Rehrauer, Hubert; Hradecky, Pavel; Gassmann, Max; Gorr, Thomas A

    2011-11-01

    Rampant activity of the hypoxia-inducible factor (HIF)-1 in cancer is frequently associated with the malignant progression into a harder-to-treat, increasingly aggressive phenotype. Clearly, anti-HIF strategies in cancer cells are of considerable clinical interest. One way to fine-tune, or inhibit, HIF's transcriptional outflow independently of hydroxylase activities could be through competing transcription factors. A CACGTG-binding activity in human hepatoma cells was previously found to restrict HIF's access to hypoxia response cis-elements (HRE) in a Daphnia globin gene promoter construct (phb2). The CACGTG factor, and its impact on hypoxia-responsive human genes, was analyzed in this study by genome-wide computational scans as well as gene-specific quantitative PCR, reporter and DNA-binding assays in hepatoma (Hep3B), cervical carcinoma (HeLa), and breast carcinoma (MCF7) cells. Among six basic helix-loop-helix transcription factors known to target CACGTG palindromes, we identified upstream stimulatory factor (USF)-1/2 as predominant phb2 CACGTG constituents in Hep3B, HeLa, and MCF7 cells. Human genes with adjacent or overlapping HRE and CACGTG motifs included with lactate dehydrogenase A (LDHA) and Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3) hypoxia-induced HIF-1 targets. Parallel recruitment of HIF-1α and USF1/2a to the respective promoter chromatin was verified for all cell lines investigated. Mutual complementing (LDHA) or moderating (BNIP3) cross-talk was seen upon overexpression or silencing of HIF-1α and USF1/2a. Distinct (LDHA) or overlapping (BNIP3) promoter-binding sites for HIF-1 and USFs were subsequently characterized. We propose that, depending on abundance or activity of its protein constituents, O(2)-independent USF signaling can function to fine-tune or interfere with HIF-mediated transcription in cancer cells. PMID:21984181

  15. Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3.

    PubMed Central

    Rietveld, L E; Holthuizen, P E; Sussenbach, J S

    1997-01-01

    Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concluded that Box A is essential for P3 activity. Box A is part of a region 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functional analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3. PMID:9581544

  16. miR-613 inhibits the growth and invasiveness of human hepatocellular carcinoma via targeting DCLK1.

    PubMed

    Wang, Wenyao; Zhang, Hongfei; Wang, Lichao; Zhang, Shaojun; Tang, Miao

    2016-05-13

    microRNAs (miRNAs) play key regulatory roles in various biological processes. In this study, we aimed to determine the expression and biological roles of miR-613 in hepatocellular carcinoma (HCC). Compared with non-cancerous liver tissues, miR-613 was significantly downregulated in HCC tissues. Ectopic expression of miR-613 significantly suppressed the proliferation and invasion of Hep3B and SMMC-7721 HCC cells. Bioinformatic and luciferase reporter analysis identified doublecortin-like kinase 1 (DCLK1) as a direct target of miR-613. Overexpression of miR-613 inhibited the expression of DCLK1 in HCC cells. There was a significant inverse correlation between miR-613 and DCLK1 protein expression in HCC samples. Small interfering RNA-mediated silencing of DCLK1 phenocopied the suppressive effects of miR-613 in HCC cells. Rescue experiments demonstrated that co-transfection of DCLK1 lacking the 3'-untranslated region partially prevented miR-613-induced suppression of HCC cell proliferation and invasion. In vivo studies confirmed that miR-613 overexpression retarded the growth of Hep3B xenograft tumors in nude mice, coupled with a reduction in the percentage of Ki67-positive tumor cells and DCLK1 protein expression. In conclusion, we provide first evidence for the suppressive activity of miR-613 in HCC, which is causally linked to targeting of DCLK1. Restoration of miR-613 may provide a potential therapeutic strategy for HCC. PMID:27049311

  17. Targeting mitogen-activated protein kinase kinase with the inhibitor PD0325901 decreases hepatocellular carcinoma growth in vitro and in mouse model systems.

    PubMed

    Hennig, Matthew; Yip-Schneider, Michele T; Wentz, Sabrina; Wu, Huangbing; Hekmatyar, S K; Klein, Patrick; Bansal, Navin; Schmidt, C Max

    2010-04-01

    Hepatocellular carcinoma (HCC) is a common cause of death from solid organ malignancy worldwide. Extracellular signal-regulated/mitogen-activated protein kinase kinase (MEK) signaling is a critical growth regulatory pathway in HCC. Targeting MEK with a novel small molecule inhibitor, PD0325901, may inhibit HCC tumorigenesis. PD0325901 (0.01-100 nM) inhibited growth and MEK activity in vitro in immortalized murine transforming growth factor alpha (TGF-alpha) transgenic hepatocyte (TAMH) cells, derived from the livers of TGF-alpha transgenic mice. Treatment of athymic mice bearing TAMH flank tumors with vehicle or PD0325901 (20 mg/kg) revealed a significant reduction of MEK activity ex vivo 24 hours after a single PD0325901 dose. The growth rate of TAMH flank tumors over 16 days was reduced threefold in the treatment arm (1113 +/- 269% versus 3077 +/- 483%, P < 0.01). PD0325901 exhibited similar inhibitory effects in HepG2 and Hep3B human HCC cells in vitro and in Hep3B flank tumors in vivo. To confirm this in a developmental model, MT-42 (CD-1) TGF-alpha mice were treated with vehicle or PD0325901 (20 mg/kg) for 5 weeks. Gross HCC was detected in 47% and 13.3% of the control and treatment mice, respectively. Tumor growth suppression by PD0325901 relative to vehicle was also shown by magnetic resonance imaging. These studies provide compelling preclinical evidence that targeting MEK in human clinical trials may be promising for the treatment of HCC. PMID:20112426

  18. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

    SciTech Connect

    Srisuttee, Ratakorn; Koh, Sang Seok; Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae; Jhun, Byung Hak; Horio, Yoshiyuki; Chung, Young-Hwa

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

  19. Expression of activins C and E induces apoptosis in human and rat hepatoma cells.

    PubMed

    Vejda, Susanne; Erlach, Natascha; Peter, Barbara; Drucker, Claudia; Rossmanith, Walter; Pohl, Jens; Schulte-Hermann, Rolf; Grusch, Michael

    2003-11-01

    Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1. PMID:12949049

  20. Live-Cell Imaging of the Association of STAT6-GFP with Mitochondria

    PubMed Central

    Khan, Rasel; Lee, Jason E.; Yang, Yang-Ming; Liang, Feng-Xia; Sehgal, Pravin B.

    2013-01-01

    The transcription factor STAT3 has been previously reported to be associated with mitochondria. However, we have been unable to visualize an association of STAT3-GFP, STAT3-DsRed or STAT3-Flag with mitochondria in human Hep3B hepatocytes thus far even though an association of these molecules with other cytoplasmic organelles (endosomes) was readily demonstrable. We then addressed the broader question of a possible association of other STAT-family of proteins with mitochondria by first using immunolocalization assays in Hep3B and human pulmonary arterial endothelial and smooth muscle cells. Strong anti-STAT6-immunolocalization with mitochondria was apparent in fluorescence and electron microscopy assays of cells first washed with a digitonin-sucrose buffer to remove bulk soluble STAT proteins. In live-cell imaging studies, STAT6-GFP, but not N1-GFP, was observed to constitutively colocalize with MitoTracker- and tetramethylrhodamine ethyl ester (TMRE)-positive mitochondria, and with mitochondrial F1-ATPase when assayed by immunofluorescence after fixation. This association was Tyr-phosphorylation independent in that a STAT6 truncated protein (STAT61-459-GFP) which lacked the SH2 domain (517–632) and the cytokine-activated Y641 phosphorylation site also accumulated in MitoTracker-positive mitochondria. This was consistent with the unexpected discovery that anti-STAT6-immunofluoresence also associated with mitochondria in mouse embryo fibroblasts (MEFs) from both wild-type and the STAT6SH2-/SH2- mouse. MEFs from the latter mouse, which had been engineered in 1996 to be deleted in the STAT6 SH2 domain (amino acids 505–584) expressed an immune-specific ∼50 kDa protein detectable in whole cell and mitochondria-enriched fractions. Taken together, the present data provide the first definitive evidence of the association of any STAT-protein family member with mitochondria - that of STAT6. PMID:23383189

  1. Inhibitor of indoleamine-2,3-dioxygenase 1-methyl-D-tryptophan can stimulate the growth of immunogenic tumors.

    PubMed

    Vasil'eva, E D; Nikolin, V P; Popova, N A; Lushnikova, E L; Kaledin, V I

    2010-10-01

    We studied the effect of 1-methyl-D-tryptophan, an inhibitor of indoleamine-2,3-dioxygenase, on the growth of transplanted hepatocarcinoma-29 in C3HA mice. Hepatocarcinoma-29 transplanted into the thigh muscles undergoes immunological rejection in more than 50% non-syngeneic recipients. Chronic local administration of 1-methyl-D-tryptophan promotes progressive growth of the tumor in recipient mice leading to 100% animal death. The stimulating effect of 1-methyl-D-tryptophan on tumor growth is discussed. PMID:21165403

  2. In vitro and in vivo antitumor efficacy of berberine-nanostructured lipid carriers against H22 tumor

    NASA Astrophysics Data System (ADS)

    Wang, Zhi-ping; Wu, Jun-biao; Chen, Tong-sheng; Zhou, Qun; Wang, Yi-fei

    2015-03-01

    Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Berberine (Ber), an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Ber loaded nanostructured lipid carriers (Ber-NLC) was prepared by hot melting and then high pressure homogenization technique. Both in vitro and in vivo anti-hepatocarcinoma effects of Ber-NLC relative to efficacy of bulk Ber were evaluated. The particle size and zeta potential of Ber-NLC were 189.3 nm and -19.3 mV, respectively. MTT assay showed that Ber-NLC effectively inhibited the proliferation of H22 cells, and the corresponding IC50 values were 6.3 μg/ml (22.1 μg/ml of bulk Ber). In vivo studies also showed higher antitumor efficacy, and inhibition rates was 68.3 % (41.4 % of bulk Ber) at 100 mg/kg intragastric administration in the H22 solid tumor bearing mice. These results suggest that the delivery of Ber-NLC is a promising approach for treating tumors.

  3. Mechanism of PAP I gene induction during hepatocarcinogenesis: clinical implications.

    PubMed Central

    Dusetti, N. J.; Montalto, G.; Ortiz, E. M.; Masciotra, L.; Dagorn, J. C.; Iovanna, J. L.

    1996-01-01

    Pancreatitis-associated protein I (PAP I) is a secretory protein first described as an acute phase reactant during acute pancreatitis. Recently, induction of the PAP I gene was also described in liver during hepatocarcinogenesis. To investigate the molecular mechanisms of this induction, we used constructs carrying progressive deletions of the PAP I promoter fused to the CAT gene. We showed that the silencer conferring tissue specificity on the PAP I gene was inactive in hepatoma cells. Then, in an vitro transcription system, we compared the transcription capacity of nuclear extracts from normal liver and HepG2 cells on constructs containing the silencer. The results confirmed that a trans-acting factor interacting with the PAP I silencer was present in liver cells and absent from hepatoma cells. On the other hand, immunohistochemistry showed that PAP I was expressed in a limited number of transformed hepatocytes. It was concluded that expression of PAP I in hepatocarcinoma occurred through inactivation of its silencer element and was not concomitant in all malignant cells. On that basis, we assayed PAP I in serum from patients with chronic hepatitis, liver cirrhosis or hepatocarcinoma. PAP I levels were normal in chronic active or persistent hepatitis, significantly higher in cirrhosis and strongly elevated in hepatocarcinoma. Because those clinical entities often develop in that sequence, serum PAP I appeared as a potential marker of hepatocarcinoma development. Images Figure 3 Figure 4 Figure 5 PMID:8956791

  4. Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

    SciTech Connect

    Li, Xiaohua; Fan, Rui; Zou, Xue; Gao, Lin; Jin, Haifeng; Du, Rui; Xia, Lin; Fan, Daiming . E-mail: fandaim@yahoo.com.cn

    2007-06-29

    Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma.

  5. In vitro antitumor efficacy of berberine: solid lipid nanoparticles against human HepG2, Huh7 and EC9706 cancer cell lines

    NASA Astrophysics Data System (ADS)

    Meng, Xiang-Ping; Wang, Xiao; Wang, Huai-ling; Chen, Tong-sheng; Wang, Yi-fei; Wang, Zhi-ping

    2016-03-01

    Hepatocarcinoma and esophageal squamous cell carcinomas threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma and esophageal carcinoma to chemotherapy. Berberine (Ber), an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma and antiesophageal carcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Ber loaded solid lipid nanoparticles (Ber-SLN) was prepared by hot melting and then high pressure homogenization technique. The in vitro anti-hepatocarcinoma and antiesophageal carcinoma effects of Ber-SLN relative to efficacy of bulk Ber were evaluated. The particle size and zeta potential of Ber-SLN were 154.3 ± 4.1 nm and -11.7 ± 1.8 mV, respectively. MTT assay showed that Ber-SLN effectively inhibited the proliferation of human HepG2 and Huh7 and EC9706 cells, and the corresponding IC50 value was 10.6 μg/ml, 5.1 μg/ml, and 7.3 μg/ml (18.3μg/ml, 6.5μg/ml, and 12.4μg/ml μg/ml of bulk Ber solution), respectively. These results suggest that the delivery of Ber-SLN is a promising approach for treating tumors.

  6. Lower concentrations of blueberry polyphenolic-rich extract differentially alter HepG2 cell proliferation and expression of genes related to cell-cycle, oxidation and epigenetic machinery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro cancer models have been used to study the effect of relatively high concentrations (>200 ug/ml) of phenolic plant extracts upon cell proliferation. In this study we report that the treatment of human hepatocarcinoma HepG2 cells with lower concentrations of blueberry phenolic extract (6.5-10...

  7. Peritoneal Dissemination of Hepatoid Carcinoma of the Ovary Treated with Cytoreductive Surgery and Hyperthermic Intraoperative Intraperitoneal Chemotherapy

    PubMed Central

    Cascales Campos, Pedro Antonio; Gil Martinez, Jose; Torroba, Amparo; Machado, Francisco; Parrila Paricio, Pascual

    2013-01-01

    Hepatoid carcinoma of the ovary (HCO) was first reported in 1987 in 5 cases of malignant ovarian tumors which were similar to hepatocarcinoma in the histological analysis. We report the first case in the literature of a patient diagnosed with HCO treated using HIPEC after extensive cytoreductive surgery, and we discuss the value of this therapeutical option in patients with HCO. PMID:23840218

  8. Predictive Relationship between Humane Values of Adolescents Cyberbullying and Cyberbullying Sensibility (Relaciones predictivas entre los valores humanos de los adolescentes, el acoso cibernético y la sensibilidad al acoso cibernético)

    ERIC Educational Resources Information Center

    Dilmac, Bülent; Yurt, Eyüp; Aydin, Mustafa; Kasarci, Ismail

    2016-01-01

    Introduction: Cyberbullying has been more common than the traditional bullying in recent years. As with the traditional bullying, humane values likely to explain the reason why adolescents tend to bully via cyber-means. Cyber-bullying behaviors also reasoned by adolescents' sensibility towards it. This study investigates the predictive…

  9. Programa Estrategico do desenvolvimento 1968-70: Area Estrategica IX. Infra-estructura Social. Educacao e Recursos Humanos, 1 e 2 (Strategic Development Program 1968-1970: Strategic Area IX. Education and Human Resources, Volumes 1 & 2).

    ERIC Educational Resources Information Center

    Brazil.

    This document is an English-language abstract (approximately 1,500 words) of a two volume work dealing with education and human resources as part of the Brazilian Government's Strategic Development Program 1968-70. It offers an integral view of education as an instrument of social transformation and an exposition of the quantitative and…

  10. miR-106b promotes cancer progression in hepatitis B virus-associated hepatocellular carcinoma

    PubMed Central

    Yen, Chia-Sheng; Su, Zhi-Ru; Lee, Yi-Ping; Liu, I-Ting; Yen, Chia-Jui

    2016-01-01

    AIM: To investigate the effect of miR-106b on tumor progression in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). METHODS: A total of 120 patients who underwent liver resection for HCC at National Cheng Kung University Hospital were enrolled in the present study. MicroRNA (miRNA) array was first used to screen the miRNA expression profiles in HCC patients. The clinical records were retrospectively analyzed, and correlations with the miRNA expression profiles were evaluated. The mRNA expression levels of the miR-106b-25 cluster (miR-106b, miR-93 and miR-25), and MCM7 in tumor and non-tumor samples were quantitated using quantitative real-time reverse transcription-polymerase chain reaction (q-RT-PCR) analysis, and correlations in the levels of miR-106b, miR-93 and miR-25 expression were calculated. Kaplan-Meier overall and disease-free survival rates of HBV-associated HCC patients were analyzed using the log-rank test based on miR-106b expression. The comparison of the miR-106b expression levels in patients with different clinical outcomes was analyzed using Mann-Whitney U tests. Furthermore, a hepatitis B virus X protein (HBx) expression plasmid was transfected into Huh7 and Hep 3B cells. The expression levels of the miR-106b-25 cluster and MCM7 in HBx-expressing Huh7 and Hep 3B cells were detected using q-RT-PCR. RESULTS: miRNA array screening showed that miR-106b and its cluster, miR-93 and miR-25 were up-regulated in HCC patients (P < 0.01). The value of miR-106b expression in HBV-associated HCC patients was significantly higher than that in HCV- (P < 0.05) or non-B/non-C- (P < 0.001) associated HCC patients. The expression of the miR-106b-25 cluster was significantly higher in tumor tissue (P < 0.001) and associated with the host gene, MCM7, in clinical specimens from HBV-associated HCC patients. Furthermore, the expression levels of miR-106b, miR-93 and miR-25 were positively correlated in HBV-associated HCC tissues (miR-106 vs miR-93, r

  11. Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma

    PubMed Central

    Wu, You-Hua; Cao, Jian-Guo; Xiang, Hong-Lin; Xia, Hong; Qin, Yong; Huang, A-Ji; Xiao, Di; Xu, Fang

    2009-01-01

    AIM: To investigate the anti-angiogenic and anti-tumor activities of recombinant vascular basement membrane-derived multifunctional peptide (rVBMDMP) in hepatocellular carcinoma (HCC). METHODS: HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen (PCNA) and CD31 immunohistochemistry. RESULTS: MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC (HepG2, Bel-7402, Hep-3B) cells and human umbilical vein endothelial (HUVE-12) cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the IC50 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil (5-FU) with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 (64.82/4.68), which was higher than that of 5-FU [SI was 1.9 (8.94/4.59)]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab (100 mg/L) did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab (100 mg/L) to HUVE-12 cells was 87.6% ± 8.2%. Alternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner. rVBMDMP (1, 3, 10 mg/kg) decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates (2.2% ± 0.73%, 4.5% ± 1.3% and 11.5% ± 3.8%) in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly higher than that (0.13% ± 0.04%) in the control group (P < 0.01). The positive

  12. The Escherichia coli Subtilase Cytotoxin A Subunit Specifically Cleaves Cell-surface GRP78 Protein and Abolishes COOH-terminal-dependent Signaling

    PubMed Central

    Ray, Rupa; de Ridder, Gustaaf G.; Eu, Jerry P.; Paton, Adrienne W.; Paton, James C.; Pizzo, Salvatore V.

    2012-01-01

    GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH2-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78low) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH2-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78. PMID:22851173

  13. Identification of a non-coding KLF4 transcript generated from intron retention and downregulated in human hepatocellular carcinoma.

    PubMed

    Zhou, Junwei; Lai, Paul Bo-San; Tsui, Stephen Kwok-Wing

    2015-10-01

    The Krüppel-like factor 4 (KLF4) gene is related to various biological processes including stem cell reprogramming and tumorigenesis. In this study, we identified and characterized a non-coding transcript of KLF4, which was designated KLF4‑003, in human liver tissue samples. KLF4‑003 was identified in a number of cell lines by reverse transcription PCR and DNA sequencing. Its expression levels were determined in 54 pairs of human hepatocellular carcinoma (HCC) tissues and a number of HCC cell lines by real-time PCR (RT-PCR). Methylation status of KLF4‑003 CpG islands was determined by bisulfite sequencing. The regulatory effect of KLF4‑003 CpG islands hypermethylation in Hep3B cells was then validated by the 5-aza-dC demethylation treatment, followed by RT-PCR analysis. Receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value for differentiating between HCC cancer and benign diseases. The association study between KLF4‑003 expression level and clinical traits of HCC patients was performed with SPSS. We found that KLF4‑003 was downregulated in 46 out of 54 HCC samples compared with their adjunct normal tissues. The reduced KLF4‑003 expression was significantly associated with HCC recurrence (P=0.045) in the follow-up of 31 HCC patients. Significant differences were detected between the methylation status of HCC specimens and their adjacent normal controls. Demethylation treatment significantly rescued the expression of KLF4‑003 in Hep3B cells. Such observation indicated that the CpG island hypermethylation was at least partially responsible for the downregulation of KLF4‑003 in HCC. The area under ROC curve for the prediction of HCC reached 0.803 (95% CI=0.719-0.886, P<0.001). Our results suggested that the expression of KLF4‑003 was epigenetically regulated by methylation status of a KLF4‑003 CpG island in HCC. The differential expression of KLF4‑003 might play an important role in HCC development and might

  14. Acteoside-mediates chemoprevention of experimental liver carcinogenesis through STAT-3 regulated oxidative stress and apoptosis.

    PubMed

    Peerzada, Kaiser J; Faridi, Aamir H; Sharma, Love; Bhardwaj, Subhash C; Satti, Naresh K; Shashi, Bhushan; Tasduq, Sheikh A

    2016-07-01

    In the absence of an effective therapy against Hepatocellular Carcinoma (HCC), chemoprevention remains an important strategy to circumvent morbidity and mortality. Here, we examined chemopreventive potential of Acteoside (ACT), a plant derived phenylethanoid glycoside against an environmental and dietary carcinogen, diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis. ACT treatment (0.1 and 0.3% supplemented with diet) started 2 weeks before DEN challenge and continued for 18 weeks thereafter, showed a remarkable chemopreventive activity. ACT treatment resulted in reduced HCC nodules. Histopathology showed progressive tissue damage, necrosis (5 weeks), hepatocytic injury (10 weeks), anisonucleosis with presence of prominent nucleoli, sinusidal dilations, and lymphomono nuclear inflammation (18 weeks). Biochemical analysis showed hepatocytic injury (raised ALT, p < 0.001), inflammation [IL-6, IFN-γ (p < 0.05), and TNF-α (p < 0.001)], apoptosis [elevated Caspase-3 (p < 0.001)]. ACT at 0.1 and 0.3% ameliorated DEN-induced pre-hepatocarcinogenic manifestations. Mechanistic studies of ACT chemoprevention was elucidated using Hep3B cells with an aim to develop an in vitro DEN-induced toxicity model. Hep3B was found to be a reliable and more sensitive towards DEN toxicity compared to HepG2 and HuH7 cells. ACT prevented DEN-induced cytotoxicity (p < 0.001), DNA damage, and genotoxicity (micronuclei test, DNA ladder test, Hoechst staining, cell cycle analysis). ACT significantly (p < 0.001) scavenged DEN-induced reactive oxygen species (ROS) levels and prevented mitochondrial membrane potential (MMP) loss. Immunoblotting showed ACT treatment reversed DEN-induced NF-κB, Bax, Cytochrome C, Bcl-2, and Stat-3 levels. We conclude that chemoprotective effect of ACT is mediated by STAT-3 dependent regulation of oxidative stress and apoptosis and ACT has potential to be developed as a chemopreventive agent. © 2015 Wiley Periodicals, Inc. Environ

  15. Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

    PubMed

    Dzieran, Johanna; Fabian, Jasmin; Feng, Teng; Coulouarn, Cédric; Ilkavets, Iryna; Kyselova, Anastasia; Breuhahn, Kai; Dooley, Steven; Meindl-Beinker, Nadja M

    2013-01-01

    Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent

  16. Bidirectional signalling between EphA2 and ephrinA1 increases tubular cell attachment, laminin secretion and modulates erythropoietin expression after renal hypoxic injury.

    PubMed

    Rodriguez, Stéphane; Rudloff, Stefan; Koenig, Katrin Franziska; Karthik, Swapna; Hoogewijs, David; Huynh-Do, Uyen

    2016-08-01

    Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular

  17. Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria.

    PubMed

    Liu, K; Shi, Y; Guo, X H; Ouyang, Y B; Wang, S S; Liu, D J; Wang, A N; Li, N; Chen, D X

    2014-01-01

    Increasing autophagy is beneficial for curing hepatocellular carcinoma (HCC). Damage-regulated autophagy modulator (DRAM) was recently reported to induce apoptosis by mediating autophagy. However, the effects of DRAM-mediated autophagy on apoptosis in HCC cells remain unclear. In this study, normal hepatocytes (7702) and HCC cell lines (HepG2, Hep3B and Huh7) were starved for 48 h. Starvation induced apoptosis and autophagy in all cell lines. We determined that starvation also induced DRAM expression and DRAM-mediated autophagy in both normal hepatocytes and HCC cells. However, DRAM-mediated autophagy was involved in apoptosis in normal hepatocytes but not in HCC cells, suggesting that DRAM-mediated autophagy fails to induce apoptosis in hepatoma in response to starvation. Immunoblot and immunofluorescence assays demonstrated that DRAM translocated to mitochondria and induced mitophagy, which led to apoptosis in 7702 cells. In HCC cells, starvation also activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which blocks the translocation of DRAM to mitochondria through the binding of p-AKT to DRAM in the cytoplasm. Inactivation of the PI3K/AKT pathway rescued DRAM translocation to mitochondria; subsequently, mitochondrial DRAM induced apoptosis in HCC cells by mediating mitophagy. Our findings open new avenues for the investigation of the mechanisms of DRAM-mediated autophagy and suggest that promoting DRAM-mediated autophagy together with PI3K/AKT inhibition might be more effective for autophagy-based therapy in hepatoma. PMID:24556693

  18. Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma.

    PubMed

    Stefanovic, Milica; Tutusaus, Anna; Martinez-Nieto, Guillermo A; Bárcena, Cristina; de Gregorio, Estefania; Moutinho, Catia; Barbero-Camps, Elisabet; Villanueva, Alberto; Colell, Anna; Marí, Montserrat; García-Ruiz, Carmen; Fernandez-Checa, Jose C; Morales, Albert

    2016-02-16

    Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated. PMID:26811497

  19. Isolation of human alpha 2HS-glycoprotein synthesized by Sf9 cells.

    PubMed

    Lörincz, Z; Kalabay, L; Cseh, S; Závodszky, P; Arnaud, P; Jakab, L

    1998-01-01

    The human alpha 2HS-glycoprotein is a negative acute phase protein synthesized by hepatocytes. Because of its fragility and the difficulty of its purification, we used recombinant DNA techniques to produce the protein in order to investigate its biological effects. The cDNA coding for the whole alpha 2HS-glycoprotein from human liver library had been cloned into the baculovirus expression vector system using the pVL 1392 transfer vector and Sf9 cells. The recombinant protein was synthesized in the Sf9 cells and was isolated on a hydroxiapatite column from the culture medium. Western blot analysis indicated that the cells synthesized large quantities of the recombinant human protein. The molecular mass of the recombinant AHSG was the same as that of the protein in human plasma but was slightly lower than that of AHSG in the cell culture supernatants of HepG2 and higher than that of AHSG from Hep3B cell cultures, respectively. PMID:9873947

  20. In vitro anti-hepatoma activity of fifteen natural medicines from Canada.

    PubMed

    Lin, Liang-Tzung; Liu, Li-Teh; Chiang, Lien-Chai; Lin, Chun-Ching

    2002-08-01

    Fifteen crude drugs, Stellaria media Cyrill. (Caryophyllaceae), Calendula officinalis L. (Compositae), Achillea millefolium L. (Compositae), Verbascum thapsus L. (Scrophulariaceae), Plantago major L. (Plantaginaceae), Borago officinalis L. (Boraginaceae), Satureja hortensis L. (Labiatae), Coptis groenlandica Salisb. (Ranunculaceae), Cassia angustifolia Vahl. (Leguminosae), Origanum majorana L. (Labiatae), Centella asiatica L. (Umbelliferae), Caulophyllum thalictroides Mich. (Berberidaceae), Picea rubens Sargent. (Pinaceae), Rhamnus purshiana D.C. (Rhamnaceae) and Hibiscus sabdariffa L. (Malvaceae), which have been used as folk medicine in Canada, were evaluated for their anti-hepatoma activity on five human liver-cancer cell lines, i.e. HepG2/C3A, SK-HEP-1, HA22T/VGH, Hep3B and PLC/PRF/5. The samples were examined by in vitro evaluation for their cytotoxicity. The results showed that the effects of crude drugs on hepatitis B virus genome-containing cell lines were different from those against non hepatitis B virus genome-containing cell lines. C. groenlandica was observed to be the most effective against the growth of all five cell lines and its chemotherapeutic values will be of interest for further studies. PMID:12203264

  1. RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis In Vitro and In Vivo

    PubMed Central

    Zhang, Xuenong; Wang, Yanyan; Han, Shishi; Xiang, Huiyao; Peng, Yan; Wu, Yinghua; Pan, Songwei; Zhang, Ye; Ruan, Jinlan

    2016-01-01

    This study aimed to investigate the anti-tumor activity of RY10-4, a small molecular that was designed and synthesized based on the structure of protoapigenone. A previous screening study showed that RY10-4 possessed anti-proliferative effects against HepG2 human hepatocellular carcinoma cells. However, the full range of RY10-4 anti-cancer effects on liver tumors and the underlying mechanisms have not been identified. Herein, employing flow cytometry, and Western blot analysis, we demonstrate that RY10-4 can induce cell cycle arrest, intracellular reactive oxygen species (ROS) production and apoptosis in HepG2 cells. In HepG2 cell xenograft tumor model, RY10-4 significantly inhibited the growth of tumors and induced apoptosis in tumor cells, with little side effects. Moreover, RY10-4 caused the suppression of STAT3 activation, which may be involved the apoptosis induction. In addition, RY10-4 inhibited the proliferation of Hep3B and HuH-7 human hepatocellular carcinoma cells in a concentration-dependent manner. Taken together, our results suggest that RY10-4 has a great potential to develop as chemotherapeutic agent for liver cancer. PMID:26974964

  2. AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2.

    PubMed

    Majumder, Rajib; Banik, Samudra Prosad; Khowala, Suman

    2016-10-01

    Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal sequencing, MALDI-TOF/MS-MS analysis and enzyme kinetics studies. AkP could cleave the growth promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 75±1.18nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration and surface proteoglycans cleavage. PMID:27180294

  3. A Review on the Synthesis and Anti-cancer Activity of 2-substituted Quinolines.

    PubMed

    Gopaul, Kaalin; Shintre, Suhas A; Koorbanally, Neil A

    2015-01-01

    Quinolines substituted at C-2 on the quinoline scaffold have shown interesting anticancer activity in a number of anticancer assays such as breast (MCF-7, MDA-MB 231), human cervical epithelioid (HeLa), oral squamous cell carcinoma (SAS), human stomach adenocarcinoma (AGS, MKN45), hepatocellular (SKHep, HepG-2, Hep-3B), prostate (PC-3, DU145), lung (A549, H-460), gastric (HGC, MNK-74), leukemia (K562, U937, REH, NALM6, CEM/ADR 5000), colon (Colo-205, HCT 116, SW620, Caco-2, HT29), neuroblastoma (IMR32), CNS (SF-268), oesophageal (EAC) and melanoma (A-375). They have been synthesised by a number of strategies starting with isatin, anilines, nitrobenzenes and benzamides and some even with cyclohexanone and cyclohexa-1,3-diones with ammonium acetate. Many of the synthetic strategies employ the derivatisation of quinoline precursors itself. We review here the synthesis of 145 bioactive anticancer quinolines substituted at the 2-position and their anticancer activity. PMID:25511516

  4. Antiangiogenic activity of 2-formyl-8-hydroxy-quinolinium chloride.

    PubMed

    Lam, K-H; Lee, K K-H; Kok, S H-L; Wong, R S-M; Lau, F-Y; Cheng, G Y-M; Wong, W-Y; Tong, S-W; Chan, K-W; Chan, R Y-K; Tang, J C-O; Cheng, C-H; Hau, D K-P; Bian, Z-X; Gambari, R; Chui, C-H

    2016-05-01

    Tumour growth is closely related to the development of new blood vessels to supply oxygen and nutrients to cancer cells. Without the neovascular formation, tumour volumes cannot increase and undergo metastasis. Antiangiogenesis is one of the most promising approaches for antitumour therapy. The exploration of new antiangiogenic agents would be helpful in antitumour therapy. Quinoline is an aromatic nitrogen compound characterized by a double-ring structure which exhibits a benzene ring fused to pyridine at two adjacent carbon atoms. The high stability of quinoline makes it preferable in a variety of therapeutic and pharmaceutical applications, including antitumour treatment. This work is to examine the potential antiangiogenic activity of the synthetic compound 2-Formyl-8-hydroxy-quinolinium chloride. We found that 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of human umbilical vein endothelial cells in vitro. Using the diethylnitrosamine-induced hepatocarcinogenesis model, 2-Formyl-8-hydroxy-quinolinium chloride showed strong antiangiogenic activity. Furthermore, 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of large Hep3B xenografted tumour from the nude mice. We assume that 2-Formyl-8-hydroxy-quinolinium chloride could be a potential antiangiogenic and antitumour agent and it is worthwhile to further study its underlying working mechanism. PMID:27133051

  5. Elimination of Cancer Stem-Like “Side Population” Cells in Hepatoma Cell Lines by Chinese Herbal Mixture “Tien-Hsien Liquid”

    PubMed Central

    Yao, Chih-Jung; Yeh, Chi-Tai; Lee, Liang-Ming; Chuang, Shuang-En; Yeh, Chuan-Feng; Chao, Wan-Ju; Lai, Tung-Yuan; Lai, Gi-Ming

    2012-01-01

    There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixture Tien-Hsien Liquid (THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such as SMO, ABCG2, CD133, β-catenin, and Oct-4 than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g., ABCG2, CD133, and SMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma. PMID:23097677

  6. Suppression of interleukin-6-induced C-reactive protein expression by FXR agonists

    SciTech Connect

    Zhang Songwen Liu Qiangyuan; Wang Juan; Harnish, Douglas C.

    2009-02-06

    C-reactive protein (CRP), a human acute-phase protein, is a risk factor for future cardiovascular events and exerts direct pro-inflammatory and pro-atherogenic properties. The farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. In this study, we report that two synthetic FXR agonists, WAY-362450 and GW4064, suppressed interleukin-6-induced CRP expression in human Hep3B hepatoma cells. Knockdown of FXR by short interfering RNA attenuated the inhibitory effect of the FXR agonists and also increased the ability of interleukin-6 to induce CRP production. Furthermore, treatment of wild type C57BL/6 mice with the FXR agonist, WAY-362450, attenuated lipopolysaccharide-induced serum amyloid P component and serum amyloid A3 mRNA levels in the liver, whereas no effect was observed in FXR knockout mice. These data provide new evidence for direct anti-inflammatory properties of FXR.

  7. Dual effects of hepatitis C virus Core protein on the transcription of cyclin-dependent kinase inhibitor p21 gene.

    PubMed

    Kwun, H J; Jang, K L

    2003-07-01

    Transcription of p21 was activated in hepatitis C virus (HCV) Core-expressing HepG2 cells where its upstream p53 was stabilized. However, this effect was not absolutely required for the activation of p21 by Core, as demonstrated in Hep3B cells. In addition, an opposite effect on the transcription of p21 was observed in NIH3T3 and primary hepatocytes, where p53 was not decreased by Core. To explain the p53-independent regulation of p21 by Core, we identified a Core-responsive element between positions -74 and -83 of the p21 promoter, exactly overlapped with a tumour growth factor beta (TGF-beta)/butyrate responsive element. Furthermore, we demonstrated that Core could activate the p21 through the element by stimulating a butyrate pathway, whereas this was inhibited through a TGF-beta pathway. The opposing effects of Core protein on the transcription of p21 might be important in understanding the progression of hepatic disease in HCV-positive patients. PMID:12823590

  8. LLL12, a novel small inhibitor targeting STAT3 for hepatocellular carcinoma therapy

    PubMed Central

    Zuo, Mingxin; Li, Chenglong; Lin, Jiayuh; Javle, Milind

    2015-01-01

    The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in clinical incidences of hepatocellular carcinoma (HCC) but not in normal human hepatocytes. STAT3 signaling plays pivotal roles in angiogenesis, survival, metastasis, and growth of HCC. Recent evidence suggests that the blockade of aberrant STAT3 pathways can be exploited as a therapeutic strategy for HCC. We have developed the novel small molecular STAT3 inhibitor LLL12 on the basis of curcumin structure using computer-aided rational design. LLL12 has shown antitumor activity in various solid tumors including breast, brain, pancreatic cancer, and glioblastoma in vitro and in vivo. In this study, we hypothesized LLL12 inhibits STAT3 phosphorylation at tyrosine 705 (Y705) in HCC and show antitumor activity in HCC in vitro and in vivo. Our results show that LLL12 selectively inhibited HCC cell proliferation and induced apoptosis in SNU387, SNU398, SNU449, and Hep3B HCC cells in vitro. Furthermore, LLL12 at 5 mg/kg/day significantly inhibited the growth of SNU398 xenografts in nude mice. Collectively, our results indicate that LLL12 could be used to target STAT3 for the effective prevention or treatment of HCC. PMID:25883212

  9. Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells

    PubMed Central

    Xie, Yuexia; Liu, Dejun; Cai, Chenlei; Chen, Xiaojing; Zhou, Yan; Wu, Liangliang; Sun, Yongwei; Dai, Huili; Kong, Xianming; Liu, Peifeng

    2016-01-01

    The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. PMID:27536098

  10. Inhibition of hardy kiwifruit (Actinidia aruguta) ripening by 1-methylcyclopropene during cold storage and anticancer properties of the fruit extract.

    PubMed

    Lim, Sooyeon; Han, Seung Hyun; Kim, Jeongyun; Lee, Han Jun; Lee, Jeong Gu; Lee, Eun Jin

    2016-01-01

    Hardy kiwifruits (Actinidia arguta) were treated with 20 μl/l 1-methylcyclopropene (1-MCP) for 16 h at 10 °C and subsequently stored at 1 ± 0.5 °C. Anticancer properties of the fruit extracts were tested against five different human cancer cells. The hardy kiwifruits, without 1-MCP treatment, showed increases in both respiration and ethylene production rates during fruit storage. The 1-MCP treatment remarkably inhibited fruit ripening by reducing respiration and ethylene production. Fruits with the 1-MCP treatment could be stored for up to 5 weeks by maintaining higher fruit firmness, ascorbic acid and total phenolic contents compared to the control. The hardy kiwifruit extracts showed anti-proliferative effects to Hep3B and HeLa cells but not to HT29, HepG2 and LoVo cells. These results suggest that the application of 1-MCP at harvest effectively delayed the ripening process of the fruits, and the fruit extract had beneficial effects for the prevention of human cancer growth. PMID:26212954

  11. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients.

    PubMed

    Serova, Maria; Tijeras-Raballand, Annemilaï; Dos Santos, Célia; Albuquerque, Miguel; Paradis, Valerie; Neuzillet, Cindy; Benhadji, Karim A; Raymond, Eric; Faivre, Sandrine; de Gramont, Armand

    2015-08-28

    Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 µM and 10 µM galunisertib, 5 µM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib. PMID:26057634

  12. Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the growth of various human cancer cell lines.

    PubMed

    Yesil-Celiktas, Ozlem; Sevimli, Canan; Bedir, Erdal; Vardar-Sukan, Fazilet

    2010-06-01

    The leaves of Rosmarinus officinalis harvested from three different locations of Turkey were extracted by both methanolic and supercritical CO(2) extraction. Subsequently, six extracts and the active compounds, carnosic acid, and rosmarinic acid were applied to various human cancer cell lines including NCI-H82 (human, small cell lung, carcinoma), DU-145 (human, prostate, carcinoma), Hep-3B (human, black, liver, carcinoma, hepatocellular), K-562 (human chronic myeloid leukemia), MCF-7 (human, breast, adenocarcinoma), PC-3 (human, prostate, adenocarcinoma) and MDA-MB-231 (human, breast, adenocarcinoma) by MTT assay. Supercritical CO(2) extracts had superior antiproliferative effect compared to the soxhlet extracts. Although the extracts exhibited various cytotoxic effects against different cell lines, comparatively low IC(50) values ranging between 12.50 and 47.55 microg/ml were attained against K-562, being the most sensitive cell line. Moreover, carnosic acid caused the lowest cell viability with values ranging from 13 to 30 % at a concentration of 19 muM after 48 h of treatments, resulting in superior antiproliferative effect. Rosemary extract is a potential candidate to be included in the anti-cancer diet with pre-determined doses avoiding toxicity. PMID:20449663

  13. Supercritical fluid extracts of rosemary leaves exhibit potent anti-inflammation and anti-tumor effects.

    PubMed

    Peng, Chiung-Huei; Su, Jeng-De; Chyau, Charng-Cherng; Sung, Tzu-Ying; Ho, Shin-Shien; Peng, Chiung-Chi; Peng, Robert Y

    2007-09-01

    Supercritical fluid SF-CO2 treatment of Rosemarinus officinalis L. fresh leaves under optimum conditions (80 degrees C at 5,000 psi) yielded 5.3% of extract supercritical fluid extraction (SFE)-80, in which five major active principles were identified by liquid chromatography/mass spectrometry (LC/MS), viz., rosmarinic acid, carnosol, 12-methoxycarnosic acid, carnosic acid, and methyl carnosate. Total phenolic content was 155.8 mg/ gallic acid equivalent (GAE)/g in SFE-80, which showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging of 81.86% at 0.01 mg/ml. When treated in RAW 264.7, apparent dose-dependent NO inhibition occurred at dosages of 1.56 to 6.25 microg/ml, and more drastically at 12.5 and 25 microg/ml. At 0.5 to 5.0 microg/ml, SFE-80 exhibited dose-dependent viability suppression and significant tumor necrosis factor alpha (TNF-alpha) production in Hep 3B, whereas no effect was found in Chang liver cells. Furthermore, no effect was observed in RAW 264.7 at dosages of 3.13 to 25 microg/ml, indicating that SFE-80 exhibited a noncytotoxic character. Conclusively, rosemary can be considered an herbal anti-inflammatory and anti-tumor agent. PMID:17827696

  14. [Synthesis and anti-proliferative activity of fluoroquinolone (rhodanine unsaturated ketone) amide derivatives].

    PubMed

    Gao, Liu-zhou; Xie, Yu-suo; Yan, Qiang; Wu, Shu-min; Ni, Li-li; Zhao, Hui; Huang, Wen-long; Hu, Guo-qiang

    2015-08-01

    To discover novel antitumor rhodanine unsaturated ketones, a series of fluoroquinolone (rhodanine α, β-unsaturated ketone) amine derivatives (5a-5r) were designed and synthesized with fluoroquinolone amide scaffold as a carrier. The structures of eighteen title compounds were characterized by elemental analysis, 1H NMR and MS. The in vitro anti-proliferative activity against Hep-3B, Capan-1 and HL60 cells was evaluated by MTT assay. The results showed that the title compounds not only had more significant anti-proliferative activity against three tested cancer cell lines than that of the parent ciprofloxacin 1, but also exhibited the highest activity against Capan-1 cells. The SAR revealed that some compounds carrying aromatic heterocyclic rings or phenyl attached to an electron-withdrawing carboxyl or sulfonamide substituent were comparable to or better than comparison doxorubicin against Capan-1 cells. As such, it suggests that fluoroquinolone (rhodanine α, β-unsaturated ketone) amines are promising leads for the development of novel antitumor fluoroquinolones or rhodanine analogues. PMID:26669001

  15. Cytotoxic triterpenoid glycosides (saikosaponins) from the roots of Bupleurum chinense.

    PubMed

    Li, Dan-Qi; Wu, Jie; Liu, Li-Yin; Wu, Ying-Ying; Li, Ling-Zhi; Huang, Xiao-Xiao; Liu, Qing-Bo; Yang, Jing-Yu; Song, Shao-Jiang; Wu, Chun-Fu

    2015-09-15

    As a part of our ongoing studies on cytotoxic triterpenoid saponins from herbal medicines, phytochemical investigation of the roots of Bupleurum chinense DC. afforded four new saikosaponins (1-4), along with 16 known ones (5-20). Their structures were established by direct interpretation of their spectral data, mainly HR-ESI-MS, 1D NMR and 2D NMR, and by comparison with literature data. Among them, compound 20 was isolated from the natural product for the first time. The cytotoxicities of all compounds against five selected human cancer cell lines (A549, HepG2, Hep3B, Bcap-37 and MCF-7) were assayed. In general, a number of the isolated compounds exhibited potent cytotoxic activities against the five selected human cancer cell lines. In particular, compounds 3, 8-9, 11-13, 16 and 20 showed more potent cytotoxic activities against the HepG2 and A549 cell lines than the positive control 5-fluorouracil. Based on the primary screening results, the preliminary structure-activity relationship (SAR) studies were also discussed. The SAR results suggest that the 13,28-epoxy bridge, the orientation of the hydroxyl group and the type of the sugar units are important requirements for cytotoxicity and selectivity. PMID:26259802

  16. Identification of NUCKS1 as a putative oncogene and immunodiagnostic marker of hepatocellular carcinoma.

    PubMed

    Cheong, Jae Youn; Kim, Young Bae; Woo, Jung Hoon; Kim, Dong Kyu; Yeo, Marie; Yang, Song-Ju; Yang, Kap-Seok; Soon, Sun Kim; Wang, Hee Jeong; Kim, Bong Wan; Park, Jun-Hyung; Cho, Sung Won

    2016-06-10

    Although the molecular mechanisms underpinning hepatocellular carcinoma (HCC) are unknown, gene copy number and associated mRNA expression changes are frequently reported. Comparative genomic hybridization arrays spotted with 4041 bacterial artificial chromosome clones were used to assess copy number changes in 45 HCC tissues. Seventy more HCC tissues were used to validate candidate genes by using western blots and immunohistochemistry. A total of 259 clones were associated with copy number changes that significantly differed between normal liver and HCC samples. The chromosomal region 1q32.1 containing the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) gene was associated with tumor vascular invasion. Western blot analysis demonstrated that NUCKS1 was up-regulated in 37 of 70 (52.8%) HCC tissues compared with adjacent non-tumor tissues, and over-expressed in a vast majority of HCCs (44/52, 84.6%) as determined by immunohistochemical staining. Furthermore, immunostaining of both NUCKS1 and glypican-3 improved the diagnostic prediction of HCC. Knock-down of NUCKS1 by siRNA implied the decrease in cell viability of the Hep3B cell line and reduced tumor formation in a xenograft mouse model. NUCKS1 was identified as a potential oncogene at chromosomal 1q32.1 in patients with HCC, and it might be a valuable immunodiagnostic marker for HCC. PMID:26968889

  17. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients

    PubMed Central

    Serova, Maria; Tijeras-Raballand, Annemilaï; Santos, Célia Dos; Albuquerque, Miguel; Paradis, Valerie; Neuzillet, Cindy; Benhadji, Karim A.; Raymond, Eric; Faivre, Sandrine; de Gramont, Armand

    2015-01-01

    Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 μM and 10 μM galunisertib, 5 μM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib. PMID:26057634

  18. The anticancer effect of chaetocin is enhanced by inhibition of autophagy.

    PubMed

    Jung, H-J; Seo, I; Casciello, F; Jacquelin, S; Lane, S W; Suh, S-Il; Suh, M-H; Lee, J S; Baek, W-K

    2016-01-01

    Chaetocin is a fungal metabolite that possesses a potent antiproliferative activity in solid tumors by inducing cell death. Although recent studies have extended the role of chaetocin in tumors, the underlying molecular mechanisms such as the downstream cascade that induces cell death has not clearly been elucidated. In this study, we show that chaetocin is able to induce both apoptosis and autophagy in several hepatoma cell lines including HepG2, Hep3B and Huh7 cell lines. Moreover, we found that the inhibition of caspase-3/7 activity by z-VAD-fmk treatment was able to block chaetocin-mediated cell death, whereas blocking autophagy by Bafilomycin A1 or the knockdown of autophagy protein 5 enhanced cell death mediated by chaetocin. These findings suggest that chaetocin has a potent anticancer effect against hepatoma. Inhibition of autophagy may potentiate anticancer effects of chaetocin thus providing evidence that combined treatment with chaetocin and autophagy inhibitors will be an effective strategy for treating cancer. PMID:26890137

  19. Nanoassemblies Based on Supramolecular Complexes of Nonionic Amphiphilic Cyclodextrin and Sorafenib as Effective Weapons to Kill Human HCC Cells.

    PubMed

    Bondì, Maria Luisa; Scala, Angela; Sortino, Giuseppe; Amore, Erika; Botto, Chiara; Azzolina, Antonina; Balasus, Daniele; Cervello, Melchiorre; Mazzaglia, Antonino

    2015-12-14

    Sorafenib (Sor), an effective chemiotherapeutic drug utilized against hepatocellular carcinoma (HCC), robustly interacts with nonionic amphiphilic cyclodextrin (aCD, SC6OH), forming, in aqueous solution, supramolecular complexes that behave as building blocks of highly water-dispersible colloidal nanoassemblies. SC6OH/Sor complex has been characterized by complementary spectroscopic techniques, such as UV-vis, steady-state fluorescence and anisotropy, resonance light scattering and (1)H NMR. The spectroscopic evidences and experiments carried out in the presence of an adamantane derivative, which competes with drug for CD cavity, agree with the entrapment of Sor in aCD, pointing out the role of the aCD cavity in the interaction between drug and amphiphile. Nanoassemblies based on SC6OH/Sor display size of ∼200 nm, negative zeta-potential (ζ = -11 mV), and both maximum loading capacity (LC ∼ 17%) and entrapment efficiency (EE ∼ 100%). Kinetic release profiles show a slower release of Sor from nanoassemblies with respect to the free drug. SC6OH/Sor nanoassemblies have very low hemolytic activity and high efficiency in vitro in decreasing cell growth and viability of HCC cell lines, such as HepG2, Hep3B, and PLC/PRF/5, opening promising chances to their in vivo applications. PMID:26528591

  20. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.

    PubMed Central

    Semenza, G L; Wang, G L

    1992-01-01

    We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis. Images PMID:1448077

  1. Chemical and biological assessment of Jujube (Ziziphus jujuba)-containing herbal decoctions: Induction of erythropoietin expression in cultures.

    PubMed

    Lam, Candy T W; Chan, Pui H; Lee, Pinky S C; Lau, Kei M; Kong, Ava Y Y; Gong, Amy G W; Xu, Miranda L; Lam, Kelly Y C; Dong, Tina T X; Lin, Huangquan; Tsim, Karl W K

    2016-07-15

    Jujubae Fructus, known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), which not only serves as daily food, but acts as tonic medicine and health supplement for blood nourishment and sedation. According to Chinese medicine, jujube is commonly included in herbal mixtures, as to prolong, enhance and harmonize the efficiency of herbal decoction, as well as to minimize the toxicity. Here, we aim to compare the chemical and pharmacological properties of three commonly used jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These decoctions share common herbs, i.e. Glycyrrhizae Radix et Rhizoma Praeparata cum Melle, Zingiberis Rhizoma Recens and Jujube, and they have the same proposed hematopoietic functions. The amount of twelve marker biomolecules deriving from different herbs in the decoctions were determined by LC-MS, and which served as parameters for chemical standardization. In general, three decoctions showed common chemical profiles but with variations in solubilities of known active ingredients. The chemical standardized decoctions were tested in cultured Hep3B cells. The herbal treatment stimulated the amount of mRNA and protein expressions of erythropoietin (EPO) via the activation of hypoxia response elements: the three herbal decoctions showed different activation. The results therefore demonstrated the hematopoietic function of decoctions and explained the enhancement of jujube function within a herbal mixture. PMID:26432380

  2. SNF5 is Involved in Suppression of Hepatocellular Carcinoma Progression via TGF-Beta 1 Signaling.

    PubMed

    Sun, Hongmiao; Zhong, Xinping; Wang, Chunyu; Wang, Shengli; Lin, Lin; Zou, Renlong; Wu, Yi; Sun, Ning; Sun, Ge; Wen, Tao; Chi, Zhi-Hong; Zhao, Yue

    2016-07-01

    SNF5 (SMARCB1/INI1/BAF47), a core subunit of SWI/SNF complex, has been reported to modulate cell proliferation and apoptosis. Genetic evidence has suggested that SNF5 participates in tumor suppression. However, the detailed biological function and underlying mechanisms of SNF5 in hepatocellular carcinoma (HCC) progression remain unclear. Here, SNF5 expression reduction in HCC tissues compared with the adjacent non-cancerous tissues has been demonstrated. Importantly, the results showed that reduced SNF5 expression has a strong correlation with worse overall survival of HCC patients. The data demonstrated that knockdown of SNF5 significantly promoted cell growth and migration in Hep3B and HCCLM3 cell lines. Interestingly, it was found that SNF5 suppressed transforming growth factor-β1 (TGF-β1) expression, and SNF5 mRNA expression was negatively correlated with TGF-β1 in HCC tissues. Furthermore, depletion of SNF5 attenuated the sensitivity of HCC cells to sorafenib. Thus, the data suggested that SNF5 may participate in HCC suppression, and reduced expression of SNF5 correlates with the poor differentiation and prognosis of HCC, indicating that SNF5 might be an important prognostic biomarker and promising therapeutic target for HCC. Anat Rec, 299:869-877, 2016. © 2016 Wiley Periodicals, Inc. PMID:27111394

  3. Tetraploidization increases sensitivity to Aurora B kinase inhibition.

    PubMed

    Marxer, Miriam; Foucar, Charles E; Man, Wing Yu; Chen, Yu; Ma, Hoi Tang; Poon, Randy Y C

    2012-07-01

    Aurora kinases are overexpressed in many cancers and are targets for anticancer drugs. The yeast homolog of Aurora B kinase, IPL1, was found to be a ploidy-specific lethality gene. Given that polyploidization is a common feature of many cancers, we hypothesized polyploidization also sensitizes mammalian cells to inhibition of Aurora kinases. Using two models of apparent diploid vs. tetraploid cell lines (one based on the hepatocellular carcinoma cell line Hep3B and another on untransformed mouse fibroblasts), we found that tetraploid cells were more sensitive to Aurora B inhibition than their diploid counterparts. Apoptosis could be induced in tetraploid cells by two different Aurora B inhibitors. Furthermore, tetraploid cells were sensitive to Aurora B inhibition but were not affected by Aurora A inhibition. Interestingly, the underlying mechanism was due to mitotic slippage and the subsequent excessive genome reduplication. In support of this, abolition of cytokinesis with dihydrocytochalasin B resulted in similar effects on tetraploid cells as Aurora B inhibition. These results indicate that inhibition of Aurora B or cytokinesis can promote apoptosis effectively in polyploid cancer cells. PMID:22722494

  4. Characterization and purification of proteins which bind high-density lipoprotein. A putative cell-surface receptor.

    PubMed Central

    Bond, H M; Morrone, G; Venuta, S; Howell, K E

    1991-01-01

    High-density lipoprotein (HDL) is shown by ligand blotting to bind membrane-associated polypeptides with sizes of 60, 100 and 210 kDa. Binding was concentration-dependent and competed by excess unlabelled HDL. All the major apolipoproteins of HDL, apoA-I, apoA-II and apoA-IV, bound independently. The 100 kDa and 210 kDa HDL-binding activities were purified from membranes of Hep3B tumour cells by ion-exchange chromatography and gel filtration. The binding activities at 100 kDa and 210 kDa co-purified. After treatment with disulphide-reducing reagent, the 210 kDa band was no longer present and an increase was observed in the amount and binding ability of the 100 kDa polypeptide. The 100 kDa binding protein labelled at the cell surface with 125I could be immunoprecipitated after cross-linking to cell-surface-bound HDL. It is proposed that this HDL-binding activity, a putative cell-surface receptor for HDL, exists totally or in part as a high-molecular-mass complex composed of 100 kDa subunits. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1659384

  5. Synthesis, characterization and biological studies of homo and hetero-binuclear 13-membered pentaaza bis (macrocyclic) complexes

    NASA Astrophysics Data System (ADS)

    Zafar, Hina; Kareem, Abdul; Sherwani, Asif; Mohammad, Owais; Khan, Tahir Ali

    2015-01-01

    A new series of homo and hetero binuclear 13-membered pentaaza bis (macrocyclic) complexes, [MM‧LX4], [M = Cu(II), M‧ = Cu(II), Co(II), Ni(II) and Sn(II); L = ligand and X = Cl or NO3] have been synthesized by the template reaction of dichloro/dinitrato diphenyl sulphone 1,3,6,9,12-tetra hydro pentaazacyclo pantane copper (II) complexes with formaldehyde, triethylenetetraamine, and respective metal salts in 1:2:1:1 molar ratio. The complexes have been characterized by elemental analyses, molar conductance measurements, ESI-mass, 1H, 13C and 119Sn NMR, IR, electronic and EPR spectral studies. The results of elemental analyses, ESI-mass and conductivity measurements confirmed the stoichiometry of the complexes while the characteristic absorption bands and resonance peaks in IR and NMR spectra confirmed the formation of macrocyclic frameworks of the complexes. These studies showed octahedral geometry around the metal ion. The thermal stability of copper complexes was also studied by TGA and DTA analyses. Some complexes of this series were also studied for their in vitro anticancer activity against cancer cells lines: Hep3B, MCF7, and HeLa. The recorded IC50 values for the tested complexes show moderate to good cytotoxicity against these cancer cell lines.

  6. Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers

    PubMed Central

    Law, Betty Yuen Kwan; Mok, Simon Wing Fai; Chan, Wai Kit; Xu, Su Wei; Wu, An Guo; Yao, Xiao Jun; Wang, Jing Rong; Liu, Liang; Wong, Vincent Kam Wai

    2016-01-01

    Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK. PMID:26811496

  7. Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells.

    PubMed

    Zheng, Wenjie; Sai, Wenli; Yao, Min; Gu, Hongbin; Yao, Yao; Qian, Qi; Yao, Dengfu

    2015-05-01

    Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of β-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells. PMID:25600802

  8. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis.

    PubMed

    Shih, Diana M; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J Mark; Vallim, Thomas; Bennett, Brian J; Graham, Mark; Hazen, Stanley L; Lusis, Aldons J

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  9. HDAC1 and HDAC2 independently predict mortality in hepatocellular carcinoma by a competing risk regression model in a Southeast Asian population

    PubMed Central

    LER, SER YENG; LEUNG, CAROL HO WING; KHIN, LAY WAI; LU, GUO-DONG; SALTO-TELLEZ, MANUEL; HARTMAN, MIKAEL; IAU, PHILIP TSAU CHOONG; YAP, CELESTIAL T.; HOOI, SHING CHUAN

    2015-01-01

    Histone deacetylases (HDACs) are enzymes involved in transcriptional repression. We aimed to examine the significance of HDAC1 and HDAC2 gene expression in the prediction of recurrence and survival in 156 patients with hepatocellular carcinoma (HCC) among a South East Asian population who underwent curative surgical resection in Singapore. We found that HDAC1 and HDAC2 were upregulated in the majority of HCC tissues. The presence of HDAC1 in tumor tissues was correlated with poor tumor differentiation. Notably, HDAC1 expression in adjacent non-tumor hepatic tissues was correlated with the presence of satellite nodules and multiple lesions, suggesting that HDAC1 upregulation within the field of HCC may contribute to tumor spread. Using competing risk regression analysis, we found that increased cancer-specific mortality was significantly associated with HDAC2 expression. Mortality was also increased with high HDAC1 expression. In the liver cancer cell lines, HEP3B, HEPG2, PLC5, and a colorectal cancer cell line, HCT116, the combined knockdown of HDAC1 and HDAC2 increased cell death and reduced cell proliferation as well as colony formation. In contrast, knockdown of either HDAC1 or HDAC2 alone had minimal effects on cell death and proliferation. Taken together, our study suggests that both HDAC1 and HDAC2 exert pro-survival effects in HCC cells, and the combination of isoform-specific HDAC inhibitors against both HDACs may be effective in targeting HCC to reduce mortality. PMID:26352599

  10. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis[S

    PubMed Central

    Shih, Diana M.; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J. Mark; Vallim, Thomas; Bennett, Brian J.; Graham, Mark; Hazen, Stanley L.; Lusis, Aldons J.

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  11. Construction of three different recombinant scorpion fusion proteins with bifunctional activity.

    PubMed

    Cui, Y; Guo, G L; Liu, Y F; Mao, Y Z; Zhang, R; Wu, C F; Zhang, J H

    2011-06-01

    This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B. PMID:21793303

  12. Quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapeutic agents.

    PubMed

    Wang, Wenge; Gallant, Jean-Nicolas; Katz, Sharyn I; Dolloff, Nathan G; Smith, Charles D; Abdulghani, Junaid; Allen, Joshua E; Dicker, David T; Hong, Bo; Navaraj, Arunasalam; El-Deiry, Wafik S

    2011-08-01

    Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10-20 mM for 1-2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3-4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo. PMID:21725212

  13. ARK5 promotes doxorubicin resistance in hepatocellular carcinoma via epithelial-mesenchymal transition.

    PubMed

    Xu, Tao; Zhang, Jian; Chen, Wei; Pan, Shengjing; Zhi, Xiao; Wen, Liang; Zhou, Yue; Chen, Bryan Wei; Qiu, Junyu; Zhang, Yun; Yang, Qi; Feng, Xinhua; Bai, Xueli; Liang, Tingbo

    2016-07-28

    AMP-activated protein kinase family member 5 (ARK5) overexpression has been reported in many human cancers, and ARK5 is associated with poor prognosis in hepatocellular carcinoma (HCC). However, whether ARK5 is involved in HCC chemoresistance is unclear. The present study aimed to investigate the role of ARK5 in HCC chemoresistance and the underlying mechanism. In this study, we found that SNU387 and SNU449 HCC cell lines overexpressing ARK5 displayed low doxorubicin sensitivity compared to Huh7 and Hep3B HCC cell lines with ARK5 low-expression. And knockdown of ARK5 increased the doxorubicin sensitivity in all HCC cell lines in the manner of inhibiting cell proliferation. Western blotting and immunofluorescence both showed that ARK5 knockdown upregulated E-cadherin and downregulated vimentin, which was consistent with the knockdown of TWIST, indicating ARK5 was involved in epithelial-mesenchymal transition (EMT). Moreover, suppressing ARK5 by siRNA restored E-cadherin and vimentin expression induced by doxorubicin treatment or hypoxia culture. Our results indicated ARK5 confers doxorubicin resistance in HCC via inducing EMT. PMID:27126361

  14. Sorafenib Enhances Radiation-Induced Apoptosis in Hepatocellular Carcinoma by Inhibiting STAT3

    SciTech Connect

    Huang, Chao-Yuan; Lin, Chen-Si; Tai, Wei-Tien; Hsieh, Chi-Ying; Shiau, Chung-Wai; Cheng, Ann-Lii; Chen, Kuen-Feng

    2013-07-01

    Purpose: Hepatocellular carcinoma (HCC) is one of the most common and lethal human malignancies. Lack of efficient therapy for advanced HCC is a pressing problem worldwide. This study aimed to determine the efficacy and mechanism of combined sorafenib and radiation therapy treatment for HCC. Methods and Materials: HCC cell lines (PLC5, Huh-7, Sk-Hep1, and Hep3B) were treated with sorafenib, radiation, or both, and apoptosis and signal transduction were analyzed. Results: All 4 HCC cell lines showed resistance to radiation-induced apoptosis; however, this resistance could be reversed in the presence of sorafenib. Inhibition of phospho-STAT3 was found in cells treated with sorafenib or sorafenib plus radiation and subsequently reduced the expression levels of STAT3-related proteins, Mcl-1, cyclin D1, and survivin. Silencing STAT3 by RNA interference overcame apoptotic resistance to radiation in HCC cells, and the ectopic expression of STAT3 in HCC cells abolished the radiosensitizing effect of sorafenib. Moreover, sorafenib plus radiation significantly suppressed PLC5 xenograft tumor growth. Conclusions: These results indicate that sorafenib sensitizes resistant HCC cells to radiation-induced apoptosis via downregulating phosphorylation of STAT3 in vitro and in vivo.

  15. The Ethanolic Extract of Taiwanofungus camphoratus (Antrodia camphorata) Induces Cell Cycle Arrest and Enhances Cytotoxicity of Cisplatin and Doxorubicin on Human Hepatocellular Carcinoma Cells

    PubMed Central

    Lin, Liang-Tzung; Tai, Chen-Jei; Su, Ching-Hua; Chang, Fang-Mo; Choong, Chen-Yen; Wang, Chien-Kai; Tai, Cheng-Jeng

    2015-01-01

    Taiwanofungus camphoratus (synonym Antrodia camphorata) is a widely used medicinal fungus in the folk medicine of Taiwan with several pharmacological features such as anti-inflammatory, liver protection, antihypertensive, and antioxidative activities. The ethanolic extract of T. camphoratus (TCEE) which contains abundant bioactive compounds including triterpenoids and polysaccharides also has antitumor effects in various human cancer cell lines. The aims of this study are to clarify the antitumor effects of TCEE on human hepatocellular carcinoma cells and also evaluate the combination drug effects with conventional chemotherapy agents, cisplatin and doxorubicin. In the present study, the TCEE treatment induced cell cycle arrest and suppressed cell growth on both Hep3B and HepJ5 cells. Expression of cell cycle inhibitors, P21 and P27, and activation of apoptosis executer enzyme, caspase-3, were also induced by TCEE. In combination with the chemotherapy agents, TCEE treatment further enhanced the tumor suppression efficiency of cisplatin and doxorubicin. These results together suggested that TCEE is a potential ingredient for developing an integrated chemotherapy for human liver cancer. PMID:26557666

  16. Supramolecular Nanotube of Chaperonin GroEL: Length Control for Cellular Uptake Using Single-Ring GroEL Mutant as End-Capper.

    PubMed

    Sim, Seunghyun; Niwa, Tatsuya; Taguchi, Hideki; Aida, Takuzo

    2016-09-01

    How to modulate supramolecular protein nanotubes without sacrificing their thermodynamic stability? This challenging issue emerged with an enhanced reality since our successful development of a protein nanotube of chaperonin GroELMC as a novel ATP-responsive 1D nanocarrier because the nanotube length may potentially affect the cellular uptake efficiency. Herein, we report a molecularly engineered protein end-capper (SRMC) that firmly binds to the nanotube termini since the end-capper originates from GroEL. According to the single-ring mutation of GroEL, we obtained a single-ring version of GroEL bearing cysteine mutations (GroELCys) and modified its 14 apical cysteine residues with merocyanine (MC). Whereas SRMC self-dimerizes upon treatment with Mg(2+), we confirmed that SRMC serves as the efficient end-capper for the Mg(2+)-mediated supramolecular polymerization of GroELMC and allows for modulating the average nanotube length over a wide range from 320 to 40 nm by increasing the feed molar ratio SRMC/GroELMC up to 5.4. We also found that the nanotubes shorter than 100 nm are efficiently taken up into HEP3B cells. PMID:27545864

  17. Selective suppression of the human aryl hydrocarbon receptor function can be mediated through binding interference at the C-terminal half of the receptor.

    PubMed

    Ren, Lina; Thompson, John D; Cheung, Michael; Ngo, Katherine; Sung, Sarah; Leong, Scott; Chan, William K

    2016-05-01

    The human aryl hydrocarbon receptor is a cytosolic signaling molecule which affects immune response and aberrant cell growth. Canonical signaling of the receptor requires the recruitment of coactivators to the promoter region to remodel local chromatin structure. We predicted that interference of this recruitment would block the aryl hydrocarbon receptor function. To prove that, we employed phage display to identify nine peptides of twelve-amino-acid in length which target the C-terminal half of the human aryl hydrocarbon receptor, including the region where coactivators bind. Eight 12mer peptides, in the form of GFP fusion, suppressed the ligand-dependent transcription of six AHR target genes (cyp1a1, cyp1a2, cyp1b1, ugt1a1, nqo1, and ahrr) in different patterns in Hep3B cells, whereas the AHR antagonist CH-223191 suppressed all these target genes similarly. Three of the 12mer peptides (namely 11-3, 1-7, and 7-3) suppressed the 3MC-induced, CYP1A1-dependent EROD activity and the ROS production caused by benzo[a]pyrene. These 12mer peptides suppressed the AHR function synergistically with CH-223191. In conclusion, we provide evidence that targeting the C-terminal half of the human aryl hydrocarbon receptor is a viable, new approach to selectively block the receptor function. PMID:26970402

  18. Antimetastatic effect of fluvastatin on breast and hepatocellular carcinoma cells in relation to SGK1 and NDRG1 genes.

    PubMed

    Salis, Osman; Okuyucu, Ali; Bedir, Abdulkerim; Gör, Ufuk; Kulcu, Canan; Yenen, Eser; Kılıç, Nermin

    2016-03-01

    Metastasis occurs due to migration of the cells from primary tumor toward other tissues by gaining invasive properties. Since metastatic invasion shows a strong resistance against conventional cancer treatments, the studies on this issue have been focused. Within this context, inhibition of migration and determination of the relationships at the gene level will contribute to treatment of metastatic cancer cases. We have aimed to demonstrate the impact of TGF-β1 and fluvastatin on human breast cancer (MCF-7) and human hepatocellular carcinoma (Hep3B) cell cultures via Real-Time Cell Analyzer (RTCA) and to test the expression levels of some genes (NDRG1, SGK1, TWIST1, AMPKA2) and to compare their gene expression levels according to RTCA results. Both of cell series were applied TGF-β1 and combinations of TGF-β1/fluvastatin. Primer and probes were synthesized using Universal Probe Library (UPL, Roche) software, and expression levels of genes were tested via qPCR using the device LightCycler 480 II (Roche). Consequently, fluvastatin dose-dependently inhibited migration induced by TGF-β1 in both groups. This inhibition was accompanied by low level of SGK1 messenger RNA (mRNA) and high levels of NDRG1 and AMPKA2 mRNA. Thus, we conclude that fluvastatin plays an important role in reducing resistance to chemotherapeutics and preventing metastasis. PMID:26419593

  19. Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human HepatocytesS⃞

    PubMed Central

    Guo, Lei; Dial, Stacey; Shi, Leming; Branham, William; Liu, Jie; Fang, Jia-Long; Green, Bridgett; Deng, Helen; Kaput, Jim

    2011-01-01

    In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites. PMID:21149542

  20. Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells.

    PubMed

    Xie, Yuexia; Liu, Dejun; Cai, Chenlei; Chen, Xiaojing; Zhou, Yan; Wu, Liangliang; Sun, Yongwei; Dai, Huili; Kong, Xianming; Liu, Peifeng

    2016-01-01

    The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. PMID:27536098

  1. R-ETODOLAC DECREASES BETA-CATENIN LEVELS ALONG WITH SURVIVAL AND PROLIFERATION OF HEPATOMA CELLS

    PubMed Central

    Behari, Jaideep; Zeng, Gang; Otruba, Wade; Thompson, Michael; Muller, Peggy; Micsenyi, Amanda; Sekhon, Sandeep S.; Leoni, Lorenzo; Monga, Satdarshan P. S.

    2007-01-01

    Background Inhibition of hepatoma cells by cyclooxygenase (COX)-2 dependent and independent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, a COX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drug Etodolac, which lacks COX-inhibitory activity, on the Wnt/β-catenin pathway and human hepatoma cells. Methods Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/β-catenin pathway components, and downstream target gene expression. Results Celecoxib at high doses affected β-catenin protein by inducing its degradation via GSK3β and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated β-catenin protein secondary to decrease in its gene expression and post-translationally through GSK3β activation. In addition, increased β-catenin-E-cadherin was also observed at the membrane. An associated inhibition of β-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutamine synthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident. Conclusion The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (at physiological doses) on HCC cells were accompanied by the down-regulation of β-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer. PMID:17275129

  2. Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma

    PubMed Central

    Stefanovic, Milica; Tutusaus, Anna; Martinez-Nieto, Guillermo A.; Bárcena, Cristina; de Gregorio, Estefania; Moutinho, Catia; Barbero-Camps, Elisabet; Villanueva, Alberto; Colell, Anna; Marí, Montserrat; García-Ruiz, Carmen; Fernandez-Checa, Jose C.; Morales, Albert

    2016-01-01

    Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated. PMID:26811497

  3. Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells.

    PubMed

    Wu, Shu-Jing; Ng, Lean-Teik; Chen, Ching-Hsein; Lin, Doung-Liang; Wang, Shyh-Shyan; Lin, Chun-Ching

    2004-03-01

    Physalis angulata and P. peruviana are herbs widely used in folk medicine. In this study, the aqueous and ethanol extracts prepared from the whole plant of these species were evaluated for their antihepatoma activity. Using XTT assay, three human hepatoma cells, namely Hep G2, Hep 3B and PLC/PRF/5 were tested. The results showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. Interestingly, all extracts showed no cytotoxic effect on normal mouse liver cells. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, a protonophore, caused a reduction of membrane potential (Deltapsim) by mitochondrial membrane depolarization. At high concentrations, EEPP was shown to induce cell cycle arrest and apoptosis through mitochondrial dysfunction as demonstrated by the following observations: (i) EEPP induced the collapse of Deltapsim and the depletion of glutathione content in a dose dependent manner; (ii) pretreatment with the antioxidant (1.0 microg/ml vitamin E) protected cells from EEPP-induced release of ROS; and (iii) at concentrations 10 to 50 microg/ml, EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) and caused G0/G1-phase arrest. Apoptosis was elicited when the cells were treated with 50 microg/ml EEPP as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. The results conclude that EEPP possesses potent antihepatoma activity and its effect on apoptosis is associated with mitochondrial dysfunction. PMID:14967200

  4. Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells.

    PubMed

    Vengellur, A; Phillips, J M; Hogenesch, J B; LaPres, J J

    2005-08-11

    Cellular, local, and organismal responses to low O2 availability occur during processes such as anaerobic metabolism and wound healing and pathological conditions such as stroke and cancer. These responses include increases in glycolytic activity, vascularization, breathing, and red blood cell production. These responses are mediated in part by the hypoxia-inducible factors (HIFs), which receive information on O2 levels from a group of iron- and O2-dependent hydroxylases. Hypoxia mimics, such as cobalt chloride, nickel chloride, and deferoxamine, act to simulate hypoxia by altering the iron status of these hydroxylases. To determine whether these mimics are appropriate substitutes for the lower O2 tension evoked naturally, we compared transcriptional responses of a Hep3B cell line using high-density oligonucleotide arrays. A battery of core genes was identified that was shared by all four treatments (hypoxia, cobalt, nickel, and deferoxamine) including glycolytic enzymes, cell cycle regulators, and apoptotic genes. Importantly, cobalt, nickel, and deferoxamine influenced transcription of distinct sets of genes that were not affected by cellular hypoxia. These global responses to hypoxia indicate a balancing act between adaptation and programmed cell death and suggest caution in the use of hypoxia mimics as substitutes for the low O2 tension that occurs in vivo. PMID:15942021

  5. Never in mitosis gene A-related kinase 6 promotes cell proliferation of hepatocellular carcinoma via cyclin B modulation

    PubMed Central

    ZHANG, BIAO; ZHANG, HAI; WANG, DONG; HAN, SHENG; WANG, KE; YAO, AIHUA; LI, XIANGCHENG

    2014-01-01

    Never in mitosis gene A-related kinase (Nek) 6 is a recently identified Nek that is required for mitotic cell cycle progression; however, the role and mechanism of Nek6 activity during hepatocarcinogenesis is not well known. The aim of this study was to investigate the potential roles and internal mechanism of Nek6 in hepatocellular carcinoma (HCC) development. In the present study, Nek6 was found to be overexpressed in HCC samples and cell lines by florescent real-time quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. Furthermore, it was evidenced to contribute to oncogenesis and progression. The ectopic overexpression of Nek6 promoted cell proliferation and colony formation, whereas gene silencing of Nek6 inhibited these phenotypes, as documented in Huh7, PLC/PRF/5, Hep3B and HepG2 HCC cell lines. Mechanistic analyses indicated that Nek6 regulates the transcription of cyclin B through cdc2 activation, and promotes the accumulation of G0/G1-phase cells. In conclusion, the findings of the current study suggested that Nek6 contributes to the oncogenic potential of HCC, and may present as a potential therapeutic target in this disease. PMID:25120679

  6. In vitro antioxidant activities and anti-proliferative properties of the functional herb Abrus cantoniensis and its main alkaloid abrine.

    PubMed

    Yang, Mei; Al Zaharna, Mazen; Chen, Yu-Shan; Li, Li; Cheung, Hon-Yeung

    2014-09-01

    Abrus cantoniensis is a common and popular vegetative food consumed as beverage, soup and folk medicine in the tropical and subtropical areas of Asia. It has been claimed valuable for cleansing toxicants in the liver. However, the functional effects of A. cantoniensis have not yet been scientifically explored. This study comprehensively evaluated the in vitro antioxidant and anti-proliferative capacities of the herbal extract and the main alkaloid abrine. Abrine was qualitatively and quantitatively determined in methanol extract (ME) using HPLC-DAD and LC-MS/MS. The results showed that ME, ethyl acetate fraction (EF) and abrine exhibited comparable ABTS radical cation scavenging activities and reducing power to two commercial antioxidants (BHT and Trolox). The EF exerted strong cellular antioxidant activity and selective cytotoxicity against three cancer cell lines in a dose-dependent manner. Biological assays revealed that the EF induced cell cycle arrest at G2/M and apoptosis in MCF-7 and Hep3B cells after 48 h of treatment. Thus, A. cantoniensis exerted potent cellular antioxidant and anti-proliferative properties, highlighting why it has been traditionally used as a functional food. PMID:25059572

  7. Combination of oncolytic adenovirus and endostatin inhibits human retinoblastoma in an in vivo mouse model.

    PubMed

    Wang, Huiping; Wei, Fang; Li, Huiming; Ji, Xunda; Li, Shuxia; Chen, Xiafang

    2013-02-01

    There is a critical need for new paradigms in retinoblastoma (RB) treatment that would more efficiently inhibit tumor growth while sparing the vision of patients. Oncolytic adenoviruses with the ability to selectively replicate and kill tumor cells are a promising strategy for cancer gene therapy. Exploration of a novel targeting strategy for RB utilizing combined oncolytic adenovirus and anti-angiogenesis therapy was applied over the course of the current study with positive results. The oncolytic adenoviruses Ad-E2F1 p-E1A and Ad-TERT p-E1 were constructed. The E1 region was regulated by the E2F-1 promoter or the human telomerase reverse transcriptase (hTERT) promoter, respectively. Effects on both replication and promotion of enhanced green fluorescent protein (EGFP) expression were observed in the replication-defective adenovirus Ad-EGFP in diverse cancer cell lines, HXO-RB44, Y79, Hep3B, NCIH460, MCF-7 and HLF. The cancer cell death induced by these agents was also explored. The in situ RB model demonstrated that mice with tumors treated with the oncolytic adenovirus and replication-defective adenovirus Ad-endostatin exhibited notable cancer cell death. This anticancer effect was further examined by stereo microscope, and the survival rate of experimental mice was determined. Both Ad-E2F1 p-E1A and Ad-TERT p-E1 replicated specifically in cancer cells in vitro and promoted EGFP expression in Ad-EGFP, although Ad-E2F1 p-E1A demonstrated superior EGFP promotion activity than Ad-TERT p-E1. In Hep3B, NCIH460 and MCF-7 cells, the number of Ad-TERT p-E1 copies was observed to exceed of the number of Ad-E2F1 p-E1A copies by a minimum of 10-fold. Furthermore, Ad-TERT p-E1 demonstrated significantly superior oncolytic effects in the RB mouse model, and Ad-endostatin effectively suppressed tumor growth and extended the overall lifespan of subjects; however, the Ad-E2F1 p-E1A was clearly less effective in attaining these goals. Most notably, the antitumor effect and

  8. RF heating of ultra-short single-walled carbon nanotubes and gadonanotubes for non-invasive cancer hyperthermia

    NASA Astrophysics Data System (ADS)

    Phounsavath, Sophia

    An emerging field of nanoparticle-mediated cancer therapy is based on the interaction of nanoparticles with radiofrequency (RF) energy to induce hyperthermia or thermal cytotoxicity within cancer cells. In this work, the heating properties of ultra-short single-walled carbon nanotubes (US-tubes) and gadonanotubes (GNTs) were assessed in an external radiofrequency field (900 W, 13.56 MHz). Surfactant (Pluronic F-108) suspensions of US-tubes (carbon-based nanocapsules that are 20--80 nm in length and 1.4 nm in diameter) and GNTs (US-tubes loaded internally with Gd3+ ions) heated in a concentration dependent manner when the RF field was applied. The observed bulk heating of the sample suspensions have been attributed to the nanomaterial itself and not the background surfactant solution. The efficacy of these remotely triggered heating agents to produce thermal cytotoxicity was then investigated in vitro in three different hepatocellular cancer cell lines (Hep3B, HepG2, and Snu449). In all cases, cancer cells that were treated with either US-tubes or GNTs in conjunction with RF had lower viabilities than those treated with RF alone. The ability of GNTs to induce thermal cytotoxicity in vivo was then investigated using subcutaneous tumor models in nude mice. Histopathological analysis of treated tumors demonstrated more pronounced and widespread cell damage in tumors treated with GNTs and RF than in the control tumors. It is anticipated that these results will aid in the future development of nanoparticle-mediated cancer therapy by hyperthermia.

  9. Growth arrest DNA damage-inducible gene 45 gamma expression as a prognostic and predictive biomarker in hepatocellular carcinoma

    PubMed Central

    Ou, Da-Liang; Shyue, Song-Kun; Lin, Liang-In; Feng, Zi-Rui; Liou, Jun-Yang; Fan, Hsiang-Hsuan; Lee, Bin-Shyun; Hsu, Chiun; Cheng, Ann-Lii

    2015-01-01

    Growth arrest DNA damage-inducible gene 45 (GADD45) family proteins play a crucial role in regulating cellular stress responses and apoptosis. The present study explored the prognostic and predictive role of GADD45γ in hepatocellular carcinoma (HCC) treatment. GADD45γ expression in HCC cells was examined using quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. The control of GADD45γ transcription was examined using a luciferase reporter assay and chromatin immunoprecipitation. The in vivo induction of GADD45γ was performed using adenoviral transfer. The expression of GADD45γ in HCC tumor tissues from patients who had undergone curative resection was measured using qRT-PCR. Sorafenib induced expression of GADD45γ mRNA and protein, independent of its RAF kinase inhibitor activity. GADD45γ induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6–7 μM) than in sorafenib-resistant HCC cells (Hep3B, Huh-7R, and HepG2R, IC50 12–15 μM). Overexpression of GADD45γ reversed sorafenib resistance in vitro and in vivo, whereas GADD45γ expression knockdown by using siRNA partially abrogated the proapoptotic effects of sorafenib on sorafenib-sensitive cells. Overexpression of survivin in HCC cells abolished the antitumor enhancement between GADD45γ overexpression and sorafenib treatment, suggesting that survivin is a crucial mediator of antitumor effects of GADD45γ. GADD45γ expression decreased in tumors from patients with HCC who had undergone curative surgery, and low GADD45γ expression was an independent prognostic factor for poor survival, in addition to old age and vascular invasion. The preceding data indicate that GADD45γ suppression is a poor prognostic factor in patients with HCC and may help predict sorafenib efficacy in HCC. PMID:26172295

  10. Evaluation of Anticancer, Antioxidant, and Possible Anti-inflammatory Properties of Selected Medicinal Plants Used in Indian Traditional Medication

    PubMed Central

    Shaikh, Rafik; Pund, Mahesh; Dawane, Ashwini; Iliyas, Sayyed

    2014-01-01

    The present study was carried out to evaluate the anticancer, antioxidant, and possible anti-inflammatory properties of diverse medicinal plants frequently used in Indian traditional medication. The selected botanicals such as Soymida fembrifuga (Roxb.) A. Juss. (Miliaceae), Tinospora cordifolia (Willd.) Miers. (Menispermaceae), Lavandula bipinnata (L.) O. Ktze. (Lamiaceae), and Helicteres isora L. (Sterculiaceae) extracted in different solvents were evaluated for their in vitro anticancer and antioxidant activities. The results obtained indicate that H. isora has potent cytotoxic activity toward the selected cancer cells such as HeLa-B75 (34.21 ± 0.24%), HL-60 (30.25 ± 1.36%), HEP-3B (25.36 ± 1.78%), and PN-15 (29.21 ± 0.52%). Interestingly, the selected botanicals selectively inhibited cyclooxygenase-2 (COX-2) more than (COX-1), which are the key enzymes implicated in inflammation. COX-2 inhibition was observed to be in the range of 19.66-49.52% as compared to COX-1 inhibition (3.93-19.61%). The results of the antioxidant study revealed that the selected plants were found to be effective 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH), and superoxide radical (SOR) scavenging agents. High-performance thin layer chromatography (HPTLC) fingerprint of flavonoids was used as a measure of quality control of the selected plant samples. The results of the present findings strengthen the potential of the selected plants as a resource for the discovery of novel anticancer, anti-inflammatory, and antioxidant agents. PMID:25379467

  11. Effects of novel neuroprotective and neurorestorative multifunctional drugs on iron chelation and glucose metabolism.

    PubMed

    Pollak, Yulia; Mechlovich, Danit; Amit, Tamar; Bar-Am, Orit; Manov, Irena; Mandel, Silvia A; Weinreb, Orly; Meyron-Holtz, Esther G; Iancu, Theodore C; Youdim, Moussa B H

    2013-01-01

    Iron accumulation and iron-related oxidative stress are involved in several pathological conditions and provide a rationale for the development of iron chelators as novel promising therapeutic strategies. Thus, we have recently synthesized multifunctional non-toxic, brain permeable iron chelating compounds, M30 and HLA20, possessing the neuroprotective N-propargyl moiety of the anti-Parkinsonian drug, monoamine oxidase (MAO)-B inhibitor, rasagiline and the antioxidant-iron chelating moiety of an 8-hydroxyquinoline derivative of the iron chelator, VK28. Here, we examined the hepatic regulatory effects of these novel compounds using two experimental approaches: chelation activity and glucose metabolism parameters. The present study demonstrated that M30 and HLA20 significantly decreased intracellular iron content and reduced ferritin expression levels in iron-loaded hepatoma Hep3B cells. In electron microscopy analysis, M30 was shown to reduce the electron-dense deposits of siderosomes by ~30 %, as well as down-regulate cytosolic ferritin particles observed in iron-overloaded cells. In vivo studies demonstrated that M30 administration (1 mg/kg, P.O. three times a week) reduced hepatic ferritin levels; increased hepatic insulin receptor and glucose transporter-1 levels and improved glucose tolerance in C57BL/6 mice and in a mouse model of type-2 diabetes, the ob/ob (leptin(-/-)). The results clearly indicate that the novel multifunctional drugs, especially M30, display significant capacity of chelating intracellular iron and regulating glucose metabolism parameters. Such effects can have therapeutic significance in conditions with abnormal local or systemic iron metabolism, including neurological diseases. PMID:22446839

  12. Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib

    PubMed Central

    Ishijima, Naoki; Kanki, Keita; Shimizu, Hiroki; Shiota, Goshi

    2015-01-01

    To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all-trans retinoic acid (ATRA), NIK-333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK-333 and Am80. We found that ATRA induced AMP-activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT-1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase-3, was observed. Suppression of AMP-activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells. PMID:25683251

  13. Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib.

    PubMed

    Ishijima, Naoki; Kanki, Keita; Shimizu, Hiroki; Shiota, Goshi

    2015-05-01

    To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all-trans retinoic acid (ATRA), NIK-333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK-333 and Am80. We found that ATRA induced AMP-activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT-1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase-3, was observed. Suppression of AMP-activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells. PMID:25683251

  14. Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism.

    PubMed

    Shah, Mehul; Patel, Kirit; Sehgal, Pravin B

    2005-04-01

    Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A "hit-and-run" type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in G2/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in Ser25 phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-1. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ("the Golgi blockade hypothesis"). PMID:15561761

  15. Programmed death ligand 1 as an indicator of pre-existing adaptive immune responses in human hepatocellular carcinoma.

    PubMed

    Xie, Qian-Kun; Zhao, Yu-Jie; Pan, Tao; Lyu, Ning; Mu, Lu-Wen; Li, Shao-Long; Shi, Mu-De; Zhang, Zhen-Feng; Zhou, Peng-Hui; Zhao, Ming

    2016-07-01

    It is well known that the aberrant expression of programmed death ligand 1 (PD-L1) on tumor cells impairs antitumor immunity. To date, in hepatocellular carcinoma (HCC), the relationship between PD-L1 expression and host-tumor immunity is not well defined. Here, the expression levels of PD-L1 and CD8(+) T cell infiltration were analyzed by immunohistochemistry (IHC) in formalin fixed paraffin embedded (FFPE) specimens from 167 HCC patients undergoing resection. A significant positive association was found between PD-L1 expression and the presence of CD8(+) T cell (p < 0.0001). Moreover, constitutive PD-L1 protein expression was not detected by western blot in HepG2, Hep3B, and 7402 HCC cancer cell lines; but co-cultured these cell lines with INFγ, a cytokine produced by activated CD8(+) T cells, remarkably upregulated PD-L1 expression. In fresh frozen HCC specimens, INFγ was found to be significantly correlated with PD-L1 and CD8(+) gene expression, as evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). These findings indicate that increased PD-L1 level may represent an adaptive immune resistance mechanism exerted by tumor cells in response to endogenous antitumor activity. Both increased intratumoral PD-L1 and CD8(+) were significantly associated with superior DFS (CD8(+): p = 0.03; PD-L1: p = 0.023) and OS (CD8(+): p = 0.001 and PD-L1: p = 0.059), but PD-L1 expression was not independently prognostic. In conclusions, PD-L1 upregulation is mainly induced by activated CD8(+) cytotoxic T cells pre-existing in HCC milieu rather than be constitutively expressed by the tumor cells, and it is a favorable prognostic factor for HCC. PMID:27622038

  16. Inhibiting the oncogenic mir-221 by microRNA sponge: toward microRNA-based therapeutics for hepatocellular carcinoma

    PubMed Central

    Moshiri, Farzaneh; Callegari, Elisa; D'Abundo, Lucilla; Corrà, Fabio; Lupini, Laura; Sabbioni, Silvia

    2014-01-01

    Aim We evaluated the capability of “microRNA sponges” in sequestering and inhibiting the over-expressed miR-221 in HCC cell lines. Background Advanced hepatocellular carcinoma (HCC) is a serious public health problem, with no effective cure at present. It has been demonstrated that the deregulation of microRNAs expression contributes to tumorigenesis. In HCC, miR-221 was shown to be up-regulated in more than 70% of the cases and was associated with higher tumor stage, metastasis and a shorter time to recurrence after surgery, suggesting an important pathogenic role. A tumor promoting function of miR-221 was proved in a transgenic mouse model, which was predisposed to the development of liver cancers. These findings suggested that miR-221 could represent a potential target for anti-tumor approaches. Material and Methods Novel adeno and adeno-associated viral vectors (AAVs) were developed: they were genetically modified to drive the expression of multiple binding sites for miR-221, the “miR-221 sponge”, which was designed to sequester miR-221 cellular molecules. Results Analysis of viral vectors activity in HCC cells revealed their capability to reduce miR-221 endogenous levels, which was accompanied by the increase in CDKN1B/ p27 protein, a known target of miR-221. An increase in apoptosis was also measured in Hep3B cells after infection with any of the two viral vectors in comparison with control vectors, with stronger effects induced by adenovirus compared to AAV vectors. Conclusion The depletion of oncogenic microRNAs represents a potential anti-cancer approach that needs to be tested for safety and efficacy. Here, we describe the development of novel “miR-221 sponge” vectors, which can reduce miR-221 activity in vitro and may be used for in vivo delivery. PMID:25436097

  17. Imaging of hepatocellular carcinoma patient-derived xenografts using 89Zr-labeled anti-glypican-3 monoclonal antibody

    PubMed Central

    Yang, Xiaoyang; Liu, Hongguang; Sun, Chris K.; Natarajan, Arutselvan; Hu, Xiang; Wang, Xiaolin; Allegretta, Mark; Guttmann, Ronald D.; Gambhir, Sanjiv S.; Chua, Mei-Sze; Cheng, Zhen; So, Samuel K.

    2015-01-01

    Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in >50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe 89Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with 89Zr, and evaluated its tumor-targeting capacity. In vitro, 89Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, 89Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, 89Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, 89Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention. PMID:24836949

  18. MicroRNA profiles in various hepatocellular carcinoma cell lines

    PubMed Central

    Morishita, Asahiro; Iwama, Hisakazu; Fujihara, Shintaro; Sakamoto, Teppei; Fujita, Koji; Tani, Joji; Miyoshi, Hisaaki; Yoneyama, Hirohito; Himoto, Takashi; Masaki, Tsutomu

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18–22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC.

  19. Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling

    PubMed Central

    Tsai, M-C; Chen, K-D; Wang, C-C; Huang, K-T; Wu, C-H; Kuo, I-Y; Chen, L-Y; Hu, T-H; Goto, S; Nakano, T; Dorling, A; McVey, J H; Chen, C-L; Lin, C-C

    2015-01-01

    We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.

  20. Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells.

    PubMed

    Teng, Yun; Radde, Brandie N; Litchfield, Lacey M; Ivanova, Margarita M; Prough, Russell A; Clark, Barbara J; Doll, Mark A; Hein, David W; Klinge, Carolyn M

    2015-06-19

    Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3-12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction. PMID:25969534

  1. Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model.

    PubMed

    Lu, Jeng-Wei; Hsia, Yu; Yang, Wan-Yu; Lin, Yu-I; Li, Chao-Chin; Tsai, Ting-Fen; Chang, Ko-Wei; Shieh, Grace S; Tsai, Shih-Feng; Wang, Horng-Dar; Yuh, Chiou-Hwa

    2012-01-01

    Hepatitis B virus X antigen plays an important role in the development of human hepatocellular carcinoma (HCC). The key regulators controlling the temporal downstream gene expression for HCC progression remains unknown. In this study, we took advantage of systems biology approach and analyzed the microarray data of the HBx transgenic mouse as a screening process to identify the differentially expressed genes and applied the software Pathway Studio to identify potential pathways and regulators involved in HCC. Using subnetwork enrichment analysis, we identified five common regulator genes: EDN1, BMP7, BMP4, SPIB and SRC. Upregulation of the common regulators was validated in the other independent HBx transgenic mouse lines. Furthermore, we verified the correlation of their RNA expression levels by using the human HCC samples, and their protein levels by using the human liver disease tissue arrays. EDN1, bone morphogenetic protein (BMP) 4 and BMP7 were upregulated in cirrhosis, BMP4, BMP7 and SRC were further upregulated in hepatocellular or cholangiocellular carcinoma samples. The trend of increasing expression of the common regulators correlates well with the progression of human liver cancer. Overexpression of the common regulators increases the cell viability, promotes migration and invasiveness and enhances the colony formation ability in Hep3B cells. Our approach allows us to identify the critical genes in hepatocarcinogenesis in an HBx-induced mouse model. The validation of the gene expressions in the liver cancer of human patients and their cellular function assays suggests that the identified common regulators may serve as useful molecular targets for the early-stage diagnosis or therapy for HCC. PMID:22021908

  2. Cinnamomum verum component 2-methoxycinnamaldehyde: a novel antiproliferative drug inducing cell death through targeting both topoisomerase I and II in human colorectal adenocarcinoma COLO 205 cells

    PubMed Central

    Tsai, Kuen-daw; Cherng, Jonathan; Liu, Yi-Heng; Chen, Ta-Wei; Wong, Ho-Yiu; Yang, Shu-mei; Chou, Kuo-Shen; Cherng, Jaw-Ming

    2016-01-01

    Background Cinnamomum verum is used to manufacture the spice cinnamon. In addition, the plant has been used as a Chinese herbal medication. Methods We investigated the antiproliferative effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the cortex of the plant, and the molecular biomarkers associated with tumorigenesis in human colorectal adenocarcinoma COLO 205 cells. Specifically, cell viability was evaluated by colorimetric assay; apoptosis was determined by flow cytometry and morphological analysis with bright field, acridine orange, and neutral red stainings, as well as comet assay; topoisomerase I activity was determined by assay based upon DNA relaxation and topoisomerase II by DNA relaxation plus decatentation of kinetoplast DNA; lysosomal vacuolation and volume of acidic compartments (VACs) were determined by neutral red staining. Results The results demonstrate that 2-MCA inhibited proliferation and induced apoptosis as implicated by mitochondrial membrane potential (ΔΨm) loss, activation of both caspase-3 and -9, increase of annexin V+PI+ cells, as well as morphological characteristics of apoptosis. Furthermore, 2-MCA also induced lysosomal vacuolation with elevated VAC, cytotoxicity, and inhibitions of topoisomerase I as well as II activities. Additional study demonstrated the antiproliferative effect of 2-MCA found in a nude mice model. Conclusions Our data implicate that the antiproliferative activity of 2-MCA in vitro involved downregulation of cell growth markers, both topoisomerase I and II, and upregulation of pro-apoptotic molecules, associated with increased lysosomal vacuolation. In vivo 2-MCA reduced the tumor burden that could have significant clinical impact. Indeed, similar effects were found in other tested cell lines, including human hepatocellular carcinoma SK-Hep-1 and Hep 3B, lung adenocarcinoma A549 and squamous cell carcinoma NCI-H520, and T-lymphoblastic MOLT-3 (results not shown). Our data implicate that 2-MCA could be a

  3. Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a μ-opioid receptor-dependent manner.

    PubMed

    Daijo, Hiroki; Kai, Shinichi; Tanaka, Tomoharu; Wakamatsu, Takuhiko; Kishimoto, Shun; Suzuki, Kengo; Harada, Hiroshi; Takabuchi, Satoshi; Adachi, Takehiko; Fukuda, Kazuhiko; Hirota, Kiichi

    2011-09-30

    Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia. PMID:21703258

  4. Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

    PubMed Central

    Rhoads, D B; Takano, M; Gattoni-Celli, S; Chen, C C; Isselbacher, K J

    1988-01-01

    The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (ii) in vivo, when rats were subjected to starvation for greater than or equal to 2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment. Images PMID:3194405

  5. Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen.

    PubMed

    Iser, David M; Warner, Nadia; Revill, Peter A; Solomon, Ajantha; Wightman, Fiona; Saleh, Suha; Crane, Megan; Cameron, Paul U; Bowden, Scott; Nguyen, Tin; Pereira, Cândida F; Desmond, Paul V; Locarnini, Stephen A; Lewin, Sharon R

    2010-06-01

    Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals. PMID:20357083

  6. Imaging of hepatocellular carcinoma patient-derived xenografts using ⁸⁹Zr-labeled anti-glypican-3 monoclonal antibody.

    PubMed

    Yang, Xiaoyang; Liu, Hongguang; Sun, Chris K; Natarajan, Arutselvan; Hu, Xiang; Wang, Xiaolin; Allegretta, Mark; Guttmann, Ronald D; Gambhir, Sanjiv S; Chua, Mei-Sze; Cheng, Zhen; So, Samuel K

    2014-08-01

    Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in >50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe (89)Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with (89)Zr, and evaluated its tumor-targeting capacity. In vitro, (89)Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, (89)Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, (89)Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, (89)Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention. PMID:24836949

  7. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  8. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

    PubMed Central

    Forsythe, J A; Jiang, B H; Iyer, N V; Agani, F; Leung, S W; Koos, R D; Semenza, G L

    1996-01-01

    Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells. PMID:8756616

  9. Assaying the reporter gene chloramphenicol acetyltransferase

    SciTech Connect

    Crabb, D.W.; Minth, C.D.; Dixon, J.E.

    1989-01-01

    These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product.

  10. Epigenetic silencing of JMJD5 promotes the proliferation of hepatocellular carcinoma cells by down-regulating the transcription of CDKN1A

    PubMed Central

    Fang, Jia-Zhu; Wu, Chong-Chao; Huang, Li-Yu; Wang, Lan; Han, Ze-Guang

    2016-01-01

    Proteins that contain jumonji C (JmjC) domains have recently been identified as major contributors to various malignant human cancers through epigenetic remodeling. However, the roles of these family members in the pathogenesis of hepatocellular carcinoma (HCC) are obscure. By mining public databases, we found that the HCC patients with lower JmjC domain-containing protein 5 (JMJD5) expression exhibited shorter survival time. We then confirmed that JMJD5 expression was indeed decreased in HCC specimens, which was caused by the altered epigenetic histone modifications, the decreased H3K9ac, H3K27ac and H3K4me2/3 together with the increased trimethylation of H3K27 and H3K9 on the JMJD5 promoter. Functional experiments revealed that JMJD5 knockdown promoted HCC cell proliferation and in vivo tumorigenicity by accelerating the G1/S transition of the cell cycle; in contrast, ectopic JMJD5 expression had the opposite effects. At molecular mechanism, we found that, in HCC cell lines including TP53-null Hep3B, JMJD5 knockdown led to the down-regulation of CDKN1A and ectopic expression of JMJD5 not only increased but also rescued CDKN1A transcription. Moreover, CDKN1A knockdown could abrogate the effect of JMJD5 knockdown or overexpression on cell proliferation, suggesting that JMJD5 inhibits HCC cell proliferation mainly by activating CDKN1A expression. We further revealed that JMJD5 directly enhances CDKN1A transcription by binding to CDKN1A's promoter independent of H3K36me2 demethylase activity. In short, we first prove that JMJD5 is a tumor suppressor gene in HCC pathogenesis, and the epigenetic silencing of JMJD5 promotes HCC cell proliferation by directly down-regulating CDKN1A transcription. PMID:26760772

  11. Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with DeltaNp73.

    PubMed

    Petitjean, A; Ruptier, C; Tribollet, V; Hautefeuille, A; Chardon, F; Cavard, C; Puisieux, A; Hainaut, P; Caron de Fromentel, C

    2008-02-01

    TP63, a member of the TP53 gene family, encodes two groups of three isoforms (alpha, beta and gamma). The TAp63 isoforms act as transcription factors. The DeltaNp63 isoforms lack the main transcription activation domain and act as dominant-negative inhibitors of transactivation (TA) isoforms. To clarify the role of these isoforms and to better understand their functional overlap with p53, we ectopically expressed each p63 isoform in the p53-null hepatocellular carcinoma cell line Hep3B. All TA isoforms, as well as DeltaNp63alpha, had a half-life of <1 h when transiently expressed and were degraded by the proteasome pathway. The most stable form was DeltaNp63gamma, with a half-life of >8 h. As expected, TA isoforms differed in their transcriptional activities toward genes regulated by p53, TAp63gamma being the most active form. In contrast, DeltaNp63 isoforms were transcriptionally inactive on genes studied and inhibited TA isoforms in a dose-dependent manner. When stably expressed in polyclonal cell populations, TAp63beta and gamma isoforms were undetectable. However, when treated with doxorubicin (DOX), p63 proteins rapidly accumulated in the cells. This stabilization was associated with an increase in phosphorylation. Strikingly, in DOX-treated polyclonal populations, increase in TAp63 levels was accompanied by overexpression of DeltaNp73. This observation suggests complex regulatory cross talks between the different isoforms of the p53 family. In conclusion, p63 exhibits several transcriptional and stress-response properties similar to those of p53, suggesting that p63 activities should be taken into consideration in approaches to improve cancer therapies based on genotoxic agents. PMID:18048390

  12. Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein.

    PubMed

    Gouas, Doriane A; Shi, Hong; Hautefeuille, Agnès H; Ortiz-Cuaran, Sandra L; Legros, Pénélope C; Szymanska, Katarzyna J; Galy, Olivier; Egevad, Lars A; Abedi-Ardekani, Behnoush; Wiman, Klas G; Hantz, Olivier; Caron de Fromentel, Claude; Chemin, Isabelle A; Hainaut, Pierre L

    2010-08-01

    Aflatoxin B(1) (AFB(1)) is a risk factor for hepatocellular carcinoma (HCC) in many low-resource countries. Although its metabolites bind at several positions in TP53, a mutation at codon 249 (AGG to AGT, arginine to serine, p.R249S) accounts for 90% of TP53 mutations in AFB(1)-related HCC. This specificity suggests that p.R249S confers a selective advantage during hepatocarcinogenesis. Using HCC cell lines, we show that p.R249S has lost the capacity to bind to p53 response elements and to transactivate p53 target genes. In p53-null Hep3B cells, stable transfection of p.R249S or of another mutant, p.R248Q, did not induce significant changes in cell proliferation and survival after cytotoxic stress. In contrast, in a cell line that constitutively expresses both p.R249S and the hepatitis B virus antigen HBx (PLC/PRF/5), silencing of either p.R249S or HBx by RNA interference slowed down proliferation, with no additive effects when both factors were silenced. Furthermore, the two proteins appear to form a complex. In human HCC samples, mutation at codon 249 did not correlate with p.R249S protein accumulation or HBx truncation status. We suggest that p.R249S may contribute to hepatocarcinogenesis through interaction with HBx, conferring a subtle growth advantage at early steps of the transformation process, but that this interaction is not required for progression to advanced HCC. PMID:20538734

  13. SW43-DOX ± loading onto drug-eluting bead, a potential new targeted drug delivery platform for systemic and locoregional cancer treatment – An in vitro evaluation

    PubMed Central

    Ludwig, Johannes M.; Gai, Yongkang; Sun, Lingyi; Xiang, Guangya; Zeng, Dexing; Kim, Hyun S.

    2016-01-01

    Treatment of unresectable primary cancer and their distant metastases, with the liver representing one of the most frequent location, is still plagued by insufficient treatment success and poor survival rates. The Sigma-2 receptor is preferentially expressed on many tumor cells making it an appealing target for therapy. Thus, we developed a potential targeted drug conjugate consisting of the Sigma-2 receptor ligand SW43 and Doxorubicin (SW43-DOX) for systemic cancer therapy and for locoregional treatment of primary and secondary liver malignancies when loaded onto drug-eluting bead (DEB) which was compared in vitro to the treatment with Doxorubicin alone. SW43-DOX binds specifically to the Sigma-2 receptor expressed on hepatocellular (Hep G2, Hep 3B), pancreatic (Panc-1) and colorectal (HT-29) carcinoma cell lines with high affinity and subsequent early specific internalization. Free SW43-DOX showed superior concentration and time depended cancer toxicity than treatment with Doxorubicin alone. Action mechanisms analysis revealed an apoptotic cell death with increased caspase 3/7 activation and reactive oxygen species (ROS) production. Only ROS scavenging with α-Tocopherol, but not the caspase inhibition (Z-VAD-FMK), partly reverted the effect. SW43-DOX could successfully be loaded onto DEB and showed prolonged eluting kinetics compared to Doxorubicin. SW43-DOX loaded DEB vs. Doxorubicin loaded DEB showed a significantly greater time dependent toxicity in all cell lines. In conclusion, the novel conjugate SW43-DOX ± loading onto DEB is a promising drug delivery platform for targeted systemic and locoregional cancer therapy. PMID:27262893

  14. Creation of a murine orthotopic hepatoma model with intra-abdominal metastasis

    PubMed Central

    Harris, Jamie; Kajdacsy-Balla, Andre; Chiu, Bill

    2016-01-01

    Aim: To create an orthotopic hepatoma model with local metastasis monitored with ultrasound could be created as a platform for testing new treatments. Background: Hepatoma accounts for 25% of liver tumors in children with poor overall survival. Intraabdominal metastasis are present in 35% of patients at time of diagnosis. We hypothesized that an orthotopic tumor model with local metastasis could be created as a platform for testing treatment modalities and could be monitored with ultrasound. Patients and methods: One million human hepatoma cells (Hep3B) were injected into the left lobe of the liver of immunocompromised mice. Tumor volume was monitored with high frequency-ultrasound until it reached 1,000mm3. At that time animals were sacrificed and examined for gross metastatic disease. Tumor sections were analyzed with hematoxylin and eosin (H&E) staining. Results: Tumor formed in 8/15 mice. The tumor was detected as small as 19.59mm3 on ultrasound. Of the forming tumors, tumor size was 145±177.93mm3 at 60 days post-injection, 665±650.39mm3 at 67 days, and reached >1000mm3 by 76.6±9.9 days. At necropsy, four mice (50%) had tumor only within the liver, four (50%) had additional tumors in omentum, pelvis and peritoneum. H&E showed tumor within the normal liver parenchyma, with multiple mitotic figures, small areas of necrosis, and hemorrhage within the tumor. Conclusion: We have successfully established an orthotopic hepatoma murine model, with a local metastatic rate of 50%. Non-invasive tumor monitoring is feasible via ultrasound. PMID:27458509

  15. Synthesis and SAR studies of novel 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives for anticancer activity

    PubMed Central

    Chen, Yi-Fong; Lin, Yi-Chien; Morris-Natschke, Susan L; Wei, Chen-Fang; Shen, Ting-Chen; Lin, Hui-Yi; Hsu, Mei-Hua; Chou, Li-Chen; Zhao, Yu; Kuo, Sheng-Chu; Lee, Kuo-Hsiung; Huang, Li-Jiau

    2015-01-01

    Background and Purpose 4-Phenylquinolin-2(1H)-one (4-PQ) derivatives can induce cancer cell apoptosis. Additional new 4-PQ analogs were investigated as more effective, less toxic antitumour agents. Experimental Approach Forty-five 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives were synthesized. Antiproliferative activities were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide assay and structure–activity relationship correlations were established. Compounds 9b, 9c, 9e and 11e were also evaluated against the National Cancer Institute-60 human cancer cell line panel. Hoechst 33258 and Annexin V-FITC/PI staining assays were used to detect apoptosis, while inhibition of microtubule polymerization was assayed by fluorescence microscopy. Effects on the cell cycle were assessed by flow cytometry and on apoptosis-related proteins (active caspase-3, -8 and -9, procaspase-3, -8, -9, PARP, Bid, Bcl-xL and Bcl-2) by Western blotting. Key Results Nine 6,7,8-substituted 4-substituted benzyloxyquinolin-2(1H)-one derivatives (7e, 8e, 9b, 9c, 9e, 10c, 10e, 11c and 11e) displayed high potency against HL-60, Hep3B, H460, and COLO 205 cancer cells (IC50 < 1 μM) without affecting Detroit 551 normal human cells (IC50 > 50 μM). Particularly, compound 11e exhibited nanomolar potency against COLO 205 cancer cells. Mechanistic studies indicated that compound 11e disrupted microtubule assembly and induced G2/M arrest, polyploidy and apoptosis via the intrinsic and extrinsic signalling pathways. Activation of JNK could play a role in TRAIL-induced COLO 205 apoptosis. Conclusion and Implications New quinolone derivatives were identified as potential pro-apoptotic agents. Compound 11e could be a promising lead compound for future antitumour agent development. PMID:25363404

  16. c9, t11- conjugated linoleic acid induces HCC cell apoptosis and correlation with PPAR-γ signaling pathway

    PubMed Central

    Lu, Guozhong; Zhang, Guoqing; Zheng, Xing; Zeng, Yan; Xu, Ziqi; Zeng, Weichi; Wang, Kebing

    2015-01-01

    Objective: Cis9, trans11 conjugated linoleic acid (c9, t11-CLA.) is one of the most important isomers of conjugated linoleic acid, which have a strong anti-tumor effects. Based on previous studies, we further explored the molecular mechanism of inducing cells apoptosis in human hepatocellular carcinoma cell line HepG2 and Hep3B. Methods: Cell Counting Kit 8 (CCK-8) assay was used to investigate the effects of c9, t11-CLA on cell viability and cell proliferation ability; The effects of c9, t11-CLA on cell apoptosis was analyzed by DNA ladder assay, immuno-fluorescence and flow cytometry, respectively. Apoptotic related gene (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bax, Bak, Bad, Bid and Bim), PPAR family member (PPAR-α, PPAR-β and PPAR-γ), and Cox2 mRNA and protein expression were analyzed by RT-PCR and western blotting. ELISA assay was used to detect the content of Caspase-3. Results: Our data were confirmed that c9, t11-CLA could inhibit the HCC cells proliferation ability and decrease the cells viability. RT-PCR and western blotting assay verified that c9, t11-CLA obviously increased the transcription and protein expression levels of PPAR-γ. The synchronism and correlation between PPAR-γ and apoptotic proteins Bcl-2, Bax and Caspase-3 were found with a dose- and time-dependent manner. PPAR-γ inhibitor GW9662 and activator Rosilitazone were further verified that there was cooperative relation between them. Conclusion: In our study, we first report that c9, t11-CLA induces apoptosis in HCC cells by activation of PPARγ-Bcl-2-Caspase-3 signal pathway. These results indicated that c9, t11-CLA will be useful for clinic therapy of anti-tumor and as a new regulator of PPAR-γ in the future. PMID:26885272

  17. Gadolinium Nanoparticles Conjugated with Therapeutic Bifunctional Chelate as a Potential T1 Theranostic Magnetic Resonance Imaging Agent.

    PubMed

    Kang, Min-Kyoung; Lee, Gang Ho; Jung, Ki-Hye; Jung, Jae-Chang; Kim, Hee-Kyung; Kim, Yeon-Hee; Lee, Jongmin; Ryeom, Hun-Kyu; Kim, Tae-Jeong; Chang, Yongmin

    2016-05-01

    This work is directed toward the synthesis of two types of gadolinium oxide nanoparticles (Gd-oxide NPs), abbreviated as Gd@SiO2-DO3A and Gd@SiO2-DO2A-BTA, with diameters of 50-60 nm. The synthesis involves sequential coating of Gd-oxide NPs with tetraethyl orthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES), followed by functionalization of the aminopropylsilane group with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid conjugates of benzothiazoles (DO3A-BTA). Gd@SiO2-DO3A and Gd@SiO2-DO2A-BTA exhibit high water solubility and colloidal stability. The r1 relaxivities of both Gd@SiO2-DO3A and Gd@SiO2-DO2A-BTA are higher than those of the corresponding low-molecular-weight magnetic resonance imaging contrast agents (MRI CAs), and their r2/r1 ratios are close to 1, indicating that both can be used as potential T1 MRI CAs. Biodistribution studies demonstrated that Gd@SiO2-DO2A-BTA was excreted via both hepatobiliary and renal pathways. Gd@SiO2-DO2A-BTA exhibits a strong intracellular uptake property in a series of tumor cell lines, and has significant anticancer characteristics against cell lines such as SK-HEP-1, MDA-MB-231, HeLa, and Hep-3B. PMID:27305813

  18. In vitro identification of nonalcoholic fatty liver disease-related protein hnRNPM

    PubMed Central

    Takino, Jun-ichi; Nagamine, Kentaro; Takeuchi, Masayoshi; Hori, Takamitsu

    2015-01-01

    AIM: To study the formation of intracellular glyceraldehyde-derived advanced glycation end products (Glycer-AGEs) in the presence of high concentrations of fructose. METHODS: Cells of the human hepatocyte cell line Hep3B were incubated with or without fructose for five days, and the corresponding cell lysates were separated by two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glycer-AGEs were detected with the anti-Glycer-AGEs antibody. Furthermore, the identification of the proteins that are modified by glyceraldehyde in the presence of high concentrations of fructose was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein and mRNA levels were determined by Western blotting and real-time reverse transcription PCR, respectively. RESULTS: The results of the two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a greater amount of Glycer-AGEs in the sample exposed to high concentrations of fructose than in the control. The detected Glycer-AGEs showed isoelectric points in the range of 8.0-9.0 and molecular weights in the range of 60-80 kDa. The heterogeneous nuclear ribonucleoprotein M (hnRNPM), which plays an important role in regulating gene expression by processing heterogeneous nuclear RNAs to form mature mRNAs, was identified as a modified protein using MALDI-TOF-MS. Increasing the concentration of fructose in the medium induced a concentration-dependent increase in the generated Glycer-AGEs. Furthermore, in an experiment using glyceraldehyde, which is a precursor of Glycer-AGEs, hnRNPM was found to be more easily glycated than the other proteins. CONCLUSION: The results suggest that glyceraldehyde-modified hnRNPM alters gene expression. This change may cause adverse effects in hepatocytes and may serve as a target for therapeutic intervention. PMID:25684943

  19. The Eltrombopag antitumor effect on hepatocellular carcinoma.

    PubMed

    Kurokawa, Tomohiro; Murata, Soichiro; Zheng, Yun-Wen; Iwasaki, Kenichi; Kohno, Keisuke; Fukunaga, Kiyoshi; Ohkohchi, Nobuhiro

    2015-11-01

    Currently, sorafenib is the only available chemotherapeutic agent for advanced hepatocellular carcinoma (HCC), but it cannot be used in patients with liver cirrhosis (LC) or thrombocytopenia. In these cases, sorafenib is likely effective if given in combination with treatments that increase the number of platelets, such as thrombopoietin (TPO) receptor agonists. Increasing the platelet count via TPO treatment resulted in reduction of LC. Eltrombopag (EP), a TPO receptor agonist, has been reported to have antitumor effects against certain cancers, despite their lack of TPO receptor expression. We hypothesized that EP may possess antitumor activity against HCC in addition to its ability to suppress hepatic fibrosis by increasing the platelet count. In the present study, the antitumor activity of EP was examined by assessing the inhibition of cell proliferation and then ascertaining the ability of iron supplementation to reverse these effects in HepG2, Hep3B and Huh7 cells. In addition, a cell cycle assay was performed using flow cytometry, and signal transduction was evaluated by analyzing cell cycle-related protein expression. The results of EP were compared with those of the most common iron chelator, deferoxamine (DFO). The combined effect of EP and sorafenib was also assessed. The results revealed that EP exerts antitumor activity in HCC that is mediated by the modulation of intracellular iron content. EP suppressed the expression of the cell cycle-related protein cyclin D1 and elicited cell cycle arrest in the G0/G1 phase. The activity of EP was comparable to that of DFO in HCC, and EP did not compete with sorafenib at low concentrations. In conclusion, our findings suggest that EP is a good candidate chemotherapeutic agent for the treatment of HCC in patients with LC and thrombocytopenia. PMID:26397763

  20. The Eltrombopag antitumor effect on hepatocellular carcinoma

    PubMed Central

    KUROKAWA, TOMOHIRO; MURATA, SOICHIRO; ZHENG, YUN-WEN; IWASAKI, KENICHI; KOHNO, KEISUKE; FUKUNAGA, KIYOSHI; OHKOHCHI, NOBUHIRO

    2015-01-01

    Currently, sorafenib is the only available chemotherapeutic agent for advanced hepatocellular carcinoma (HCC), but it cannot be used in patients with liver cirrhosis (LC) or thrombocytopenia. In these cases, sorafenib is likely effective if given in combination with treatments that increase the number of platelets, such as thrombopoietin (TPO) receptor agonists. Increasing the platelet count via TPO treatment resulted in reduction of LC. Eltrombopag (EP), a TPO receptor agonist, has been reported to have antitumor effects against certain cancers, despite their lack of TPO receptor expression. We hypothesized that EP may possess antitumor activity against HCC in addition to its ability to suppress hepatic fibrosis by increasing the platelet count. In the present study, the antitumor activity of EP was examined by assessing the inhibition of cell proliferation and then ascertaining the ability of iron supplementation to reverse these effects in HepG2, Hep3B and Huh7 cells. In addition, a cell cycle assay was performed using flow cytometry, and signal transduction was evaluated by analyzing cell cycle-related protein expression. The results of EP were compared with those of the most common iron chelator, deferoxamine (DFO). The combined effect of EP and sorafenib was also assessed. The results revealed that EP exerts antitumor activity in HCC that is mediated by the modulation of intracellular iron content. EP suppressed the expression of the cell cycle-related protein cyclin D1 and elicited cell cycle arrest in the G0/G1 phase. The activity of EP was comparable to that of DFO in HCC, and EP did not compete with sorafenib at low concentrations. In conclusion, our findings suggest that EP is a good candidate chemotherapeutic agent for the treatment of HCC in patients with LC and thrombocytopenia. PMID:26397763

  1. Effects of Sorafenib Dose on Acquired Reversible Resistance and Toxicity in Hepatocellular Carcinoma.

    PubMed

    Kuczynski, Elizabeth A; Lee, Christina R; Man, Shan; Chen, Eric; Kerbel, Robert S

    2015-06-15

    Acquired evasive resistance is a major limitation of hepatocellular carcinoma (HCC) treatment with the tyrosine kinase inhibitor (TKI) sorafenib. Recent findings suggest that resistance to sorafenib may have a reversible phenotype. In addition, loss of responsiveness has been proposed to be due to a gradual decrease in sorafenib plasma levels in patients. Here, the possible mechanisms underlying reversible sorafenib resistance were investigated using a Hep3B-hCG orthotopic human xenograft model of locally advanced HCC. Tissue and plasma sorafenib and metabolite levels, downstream antitumor targets, and toxicity were assessed during standard and dose-escalated sorafenib treatment. Drug levels were found to decline significantly over time in mice treated with 30 mg/kg sorafenib, coinciding with the onset of resistance but a greater magnitude of change was observed in tissues compared with plasma. Skin rash also correlated with drug levels and tended to decrease in severity over time. Drug level changes appeared to be partially tumor dependent involving induction of tumoral CYP3A4 metabolism, with host pretreatment alone unable to generate resistance. Escalation from 30 to 60 mg/kg sorafenib improved antitumor efficacy but worsened survival due to excessive body weight loss. Microvessel density was inhibited by sorafenib treatment but remained suppressed over time and dose increase. In conclusion, tumor CYP3A4 induction by sorafenib is a novel mechanism to account for variability in systemic drug levels; however, declining systemic sorafenib levels may only be a minor resistance mechanism. Escalating the dose may be an effective treatment strategy, provided toxicity can be controlled. PMID:25908587

  2. Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

    PubMed

    Suzuki, Takehiro; Yamaguchi, Hiroaki; Kikusato, Motoi; Hashizume, Osamu; Nagatoishi, Satoru; Matsuo, Akihiro; Sato, Takeya; Kudo, Tai; Matsuhashi, Tetsuro; Murayama, Kazutaka; Ohba, Yuki; Watanabe, Shun; Kanno, Shin-Ichiro; Minaki, Daichi; Saigusa, Daisuke; Shinbo, Hiroko; Mori, Nobuyoshi; Yuri, Akinori; Yokoro, Miyuki; Mishima, Eikan; Shima, Hisato; Akiyama, Yasutoshi; Takeuchi, Yoichi; Kikuchi, Koichi; Toyohara, Takafumi; Suzuki, Chitose; Ichimura, Takaharu; Anzai, Jun-Ichi; Kohzuki, Masahiro; Mano, Nariyasu; Kure, Shigeo; Yanagisawa, Teruyuki; Tomioka, Yoshihisa; Toyomizu, Masaaki; Tsumoto, Kohei; Nakada, Kazuto; Bonventre, Joseph V; Ito, Sadayoshi; Osaka, Hitoshi; Hayashi, Ken-Ichi; Abe, Takaaki

    2016-07-01

    Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction. PMID:26609120

  3. Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines

    SciTech Connect

    Di Fazio, Pietro; Montalbano, Roberta; Neureiter, Daniel; Alinger, Beate; Schmidt, Ansgar; Merkel, Anna Lena; Quint, Karl; Ocker, Matthias

    2012-09-10

    Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate. -- Highlights: Black-Right-Pointing-Pointer Panobinostat for the treatment of liver cancer. Black-Right-Pointing-Pointer Panobinostat meddles with miRNAs-dependent transcriptional and translational control. Black-Right-Pointing-Pointer Tumorsuppressor miRNA hsa-let-7b upregulation. Black-Right-Pointing-Pointer HMGA2 is downregulated via RNA interference pathways mediated by hsa-let-7b. Black-Right-Pointing-Pointer Panobinostat determines inhibition of

  4. Interleukin-12 inhibits the hepatocellular carcinoma growth by inducing macrophage polarization to the M1-like phenotype through downregulation of Stat-3.

    PubMed

    Wang, Qin; Cheng, Feng; Ma, Ting-Ting; Xiong, Hai-Yu; Li, Zi-Wei; Xie, Chang-Li; Liu, Cui-Ying; Tu, Zhi-Guang

    2016-04-01

    Hepatocellular carcinoma is the third most common cause of cancer death worldwide. Novel early detection biomarkers and efficacious therapy strategies are needed. Macrophages recruited from circulation monocytes are the major component of solid cancer and play an important role in the carcinogenesis. Whether overexpression of L-12 in monocytes could induce the phenotype directional differentiation into tumoricidal M1 macrophages and inhibit HCC growth in tumor microenvironment was investigated in this study. For the establishment of the monocyte/IL-12 and polarization of M1-like macrophage, the IL-12 overexpressing recombinant monocyte/IL-12 cells were established by infecting with pAd5F35-CMV/IL-12 adenovirus and co-cultured with HCC SMMC-7721 and Hep3B cells. It was found that the phenotype of monocyte/IL-12 polarized to M1-like macrophages with CD197high IL-12high CD206low IL-10low, and decreased expression of TGF-β, VEGF-A, and MMP-9. In order to explore the mechanism underlying the macrophages polarization, we detected the Stat-3 pathway and its downstream transcription factor c-myc, and found that the p-Stat-3 and c-myc were down-regulated. To evaluate the effects of monocyte/IL-12 on inhibiting HCC growth, various assays including CCK8, flow cytometry, colony-forming and Transwell assays in vitro, and xenograft mouse models and immunohistochemical analyses in vivo were used to detect the HCC growth and relative markers. Treated with IL-12 overexpressing monocytes, the xenograft tumor growth was significantly inhibited in vivo. These results have proven that IL-12-overexpressed monocytes could directionally differentiate to M1-like macrophages through downregulation of Stat-3 and result in the inhibition of HCC growth. PMID:27003285

  5. The Transcriptional Profiling of Glycogenes Associated with Hepatocellular Carcinoma Metastasis

    PubMed Central

    Liu, Tianhua; Zhang, Shu; Chen, Jie; Jiang, Kai; Zhang, Qinle; Guo, Kun; Liu, Yinkun

    2014-01-01

    Background and objective Metastasis is one of the important reasons for the poor prognosis of hepatocellular carcinoma (HCC), abnormal glycosylation plays a pivotal role in HCC metastasis. The goal of this study was to screen and validate the transcriptional profiling of glycogenes associated with HCC metastasis. Methodology The differentially transcribed glycogenes were screened out by the Human Glycosylation RT2 Profiler PCR Array, and were identified by qRT-PCR in human HCC cell lines and their orthotopic xenograft tumors. Further analyses were performed with K-mean clustering, Gene Ontology (GO) and ingenuity pathways analysis (IPA). Four differentially transcribed glycogenes were validated in clinical cancer specimens by qRT-PCR. Results A total of thirty-three differentially transcribed glycogenes were obtained by comparison the transcription in the metastatic human HCC cell lines (MHCC97L, MHCC97H and HCCLM3) with the transcription in the non-metastatic HCC cell line Hep3B. Seven differentially transcribed glycogenes were selected to further identification in human HCC cell lines and their orthotopic xenograft tumors. According to their trends by K-mean clustering, all of the differentially transcribed glycogenes were classified in six clusters. GO analysis of the differentially transcribed glycogenes described them in biological process, subcellular location and molecular function. Furthermore, the partial regulatory network of the differentially transcribed glycogenes was acquired through the IPA. The transcription levels of galnt3, gcnt3, man1a1, mgat5b in non-metastatic and metastatic HCC clinical cancer specimens showed the same changing trends with the results in human HCC cell lines and their orthotopic xenograft tumors, and the divergent transcription levels of gcnt3 and mgat5b were statistically significant. Conclusions The transcriptional profiling of glycogenes associated with HCC metastasis was obtained and validated in this study and it might

  6. Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation.

    PubMed

    Okuyama, Noriko; Ide, Yoshihito; Nakano, Miyako; Nakagawa, Tsutomu; Yamanaka, Kanako; Moriwaki, Kenta; Murata, Kohei; Ohigashi, Hiroaki; Yokoyama, Shigekazu; Eguchi, Hidetoshi; Ishikawa, Osamu; Ito, Toshifumi; Kato, Michio; Kasahara, Akinori; Kawano, Sunao; Gu, Jianguo; Taniguchi, Naoyuki; Miyoshi, Eiji

    2006-06-01

    Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An approximately 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the beta chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the alpha 1-3/alpha 1-4/alpha 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver. PMID:16385567

  7. Impact of Metronomic UFT/Cyclophosphamide Chemotherapy and Antiangiogenic Drug Assessed in a New Preclinical Model of Locally Advanced Orthotopic Hepatocellular Carcinoma1

    PubMed Central

    Tang, Terence C; Man, Shan; Lee, Christina R; Xu, Ping; Kerbel, Robert S

    2010-01-01

    Hepatocellular carcinoma (HCC) is an intrinsically chemotherapy refractory malignancy. Development of effective therapeutic regimens would be facilitated by improved preclinical HCC models. Currently, most models consist of subcutaneous human tumor transplants in immunodeficient mice; however, these do not reproduce the extensive liver disease associated with HCC or metastasize. To address this deficiency, we developed an orthotopic model. Human HCC cells were transfected with the gene encoding secretable β-subunit human choriogonadotropin (β-hCG), which was used as a surrogate marker of tumor burden. The HCC cells were implanted into the left liver lobe of severe combined immunodeficient (SCID) mice, after which the efficacy of different therapies was evaluated on established, but liver-confined human Hep3B cell line HCC. Treatments included sorafenib or metronomic chemotherapy using cyclophosphamide (CTX), UFT, an oral 5-fluorouracil prodrug, or doxorubicin either alone or in various combinations, with or without an antiangiogenic agent, DC101, an anti-vascular endothelial growth factor receptor-2 antibody. Sorafenib inhibited tumor growth in a dose-dependent manner but caused severe weight loss in SCID mice, thus necessitating use of DC101 in subsequent experiments. Although less toxicity was observed using either single or doublet metronomic chemotherapy without any added antiangiogenic agent, none, provided survival benefit. In contrast, significantly improved overall survival was observed using various combinations of metronomic chemotherapy regimens such as UFT + CTX with DC101. In conclusion, using this model of liver-confined but advanced HCC suggests that the efficacy of a targeted antiangiogenic drug or metronomic chemotherapy can be mutually enhanced by concurrent combination treatment. PMID:20234820

  8. Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen induction by hypoxia and hypoxia-inducible factors.

    PubMed

    Veeranna, Ravindra P; Haque, Muzammel; Davis, David A; Yang, Min; Yarchoan, Robert

    2012-01-01

    Hypoxia and hypoxia-inducible factors (HIFs) play an important role in the Kaposi's sarcoma-associated herpesvirus (KSHV) life cycle. In particular, hypoxia can activate lytic replication of KSHV and specific lytic genes, including the replication and transcription activator (RTA), while KSHV infection in turn can increase the levels and activity of HIFs. In the present study, we show that hypoxia increases the levels of mRNAs encoding KSHV latency-associated nuclear antigen (LANA) in primary effusion lymphoma (PEL) cell lines and also increases the levels of LANA protein. Luciferase reporter assays in Hep3B cells revealed a moderate activation of the LANA promoter region by hypoxia as well as by cotransfection with degradation-resistant HIF-1α or HIF-2α expression plasmids. Computer analysis of a 1.2-kb sequence upstream of the LANA translational start site identified six potential hypoxia-responsive elements (HRE). Sequential deletion studies revealed that much of this activity was mediated by one of these HREs (HRE 4R) oriented in the 3' to 5' direction and located between the constitutive (LTc) and RTA-inducible (LTi) mRNA start sites. Site-directed mutation of this HRE substantially reduced the response to both HIF-1α and HIF-2α in a luciferase reporter assay. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated binding of both HIF-1α and HIF-2α to this region. Also, HIF-1α was found to associate with RTA, and HIFs enhanced the activation of LTi by RTA. These results provide evidence that hypoxia and HIFs upregulate both latent and lytic KSHV replication and play a central role in the life cycle of this virus. PMID:22090111

  9. Ethanolic Extract of Agaricus blazei Fermentation Product Inhibits the Growth and Invasion of Human Hepatoma HA22T/VGH and SK-Hep-1 Cells

    PubMed Central

    Tung, Yen-Chen; Su, Zheng-Yuan; Kuo, Min-Liang; Sheen, Lee-Yan

    2012-01-01

    Hepatoma is a leading cause of death in the world. SK-Hep-1 and HA22T/VGH cells are poorly differentiated human hepatocellular carcinoma cell lines with invasive and migratory abilities. Agaricus blazei (AB) is a mushroom with many biological effects and active ingredients, and the ethanolic extract of AB fermentation product (AB-pE) was demonstrated to inhibit the growth of hepatoma Hep3B and HepG2 cells in our previous study. In this study, we further investigated the anticancer and anti-invasive abctivities of the AB-pE. Results showed that the AB-pE inhibited the growth of SK-Hep1 and HA22T/VGH cells (with IC50 values of 26.8 and 28.7 μg/mL, respectively) and led cells toward apoptosis after 48 h of treatment. Activation of caspase-3 by AB-pE (12.5~200 μg/mL) in a dose-dependent manner was observed in both cell lines using fluorescence microscopy and flow cytometry. The apoptosis triggered by the AB-pE was regulated by the increased expression of Bax, the activation of caspase-3, caspase-9, and PARP, and the decreased expression of Bcl-2. Additionally, the AB-pE showed the potential ability to inhibit invasion of SK-Hep1 and HA22T/VGH cells according to the results of a Matrigel invasion assay. Our results suggested that the AB-pE may be a further developed for its potential against hepatoma due to its antiproliferative (via apoptosis) and anti-invasive activities in hepatoma cells. PMID:24716127

  10. Discovery of a novel anticancer agent with both anti-topoisomerase I and II activities in hepatocellular carcinoma SK-Hep-1 cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde

    PubMed Central

    Perng, Daw-Shyong; Tsai, Yu-Hsin; Cherng, Jonathan; Wang, Jeng-Shing; Chou, Kuo-Shen; Shih, Chia-Wen; Cherng, Jaw-Ming

    2016-01-01

    Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine for various applications. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human hepatocellular carcinoma SK-Hep-1 cell line. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase-3 and caspase-9, increase in the DNA content in sub-G1, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments, suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2, prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against SK-Hep-1 cells is accompanied by downregulations of NF-κB-binding activity, inflammatory responses involving cyclooxygenase-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and volume of acidic compartments. Similar effects (including all of the above-mentioned effects) were found in other tested cell lines, including human hepatocellular carcinoma Hep 3B, lung adenocarcinoma A549, squamous cell carcinoma NCI-H520, colorectal adenocarcinoma COLO 205, and T-lymphoblastic MOLT-3 (results not shown). Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26792981

  11. Discovery of a novel anticancer agent with both anti-topoisomerase I and II activities in hepatocellular carcinoma SK-Hep-1 cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

    PubMed

    Perng, Daw-Shyong; Tsai, Yu-Hsin; Cherng, Jonathan; Wang, Jeng-Shing; Chou, Kuo-Shen; Shih, Chia-Wen; Cherng, Jaw-Ming

    2016-01-01

    Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine for various applications. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human hepatocellular carcinoma SK-Hep-1 cell line. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase-3 and caspase-9, increase in the DNA content in sub-G1, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments, suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2, prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against SK-Hep-1 cells is accompanied by downregulations of NF-κB-binding activity, inflammatory responses involving cyclooxygenase-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and volume of acidic compartments. Similar effects (including all of the above-mentioned effects) were found in other tested cell lines, including human hepatocellular carcinoma Hep 3B, lung adenocarcinoma A549, squamous cell carcinoma NCI-H520, colorectal adenocarcinoma COLO 205, and T-lymphoblastic MOLT-3 (results not shown). Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26792981

  12. Cytotoxic effects of new geranyl chalcone derivatives isolated from the leaves of Artocarpus communis in SW 872 human liposarcoma cells.

    PubMed

    Fang, Song-Chwan; Hsu, Chin-Lin; Yu, Yu-Shen; Yen, Gow-Chin

    2008-10-01

    Breadfruit (Artocarpus communis Moraceae) is cultivated in tropical and subtropical regions as a traditional starch crop and also has potential medicinal properties. The aim of this work was to study the in vitro anticancer activity of compounds isolated from the leaves of Artocarpus communis. Three new geranyl chalcone derivatives including isolespeol (1), 5'-geranyl-2',4',4-trihydroxychalcone (2), and 3,4,2',4'-tetrahydroxy-3'-geranyldihydrochalcone (3), together with two known compounds lespeol (4) and xanthoangelol (5), were isolated from the leaves of Artocarpus communis. The structures of 1- 5 were elucidated by spectroscopy and through comparison with data reported in the literature. The effects of geranyl chalcone derivatives (1- 5) on the viability of human cancer cells (including SW 872, HT-29, COLO 205, Hep3B, PLC5, Huh7, and HepG2 cells) were investigated. The results indicate that isolespeol (1) showed the highest inhibitory activity with an IC 50 value of 3.8 muM in SW 872 human liposarcoma cells. Treatment of SW 872 human liposarcoma cells with isolespeol (1) caused the loss of mitochondrial membrane potential (DeltaPsim). Western blotting revealed that isolespeol (1) stimulated increased protein expression of Fas, FasL, and p53. The expression ratios of pro- and antiapoptotic Bcl-2 family members were also changed by isolespeol (1) treatment to subsequently induce the activation of caspase-9 and caspase-3, which was followed by cleavage of poly (ADP-ribose) polymerase (PARP). These results demonstrate that isolespeol (1) induces apoptosis in SW 872 cells through Fas- and mitochondria-mediated pathways. PMID:18767861

  13. Sensitive multiplex detection of serological liver cancer biomarkers using SERS-active photonic crystal fiber probe.

    PubMed

    Dinish, U S; Balasundaram, Ghayathri; Chang, Young Tae; Olivo, Malini

    2014-11-01

    Surface-enhanced Raman scattering (SERS) spectroscopy possesses the most promising advantage of multiplex detection for biosensing applications, which is achieved due to the narrow 'fingerprint' Raman spectra from the analyte molecules. We developed an ultrasensitive platform for the multiplex detection of cancer biomarkers by combining the SERS technique with a hollow-core photonic crystal fiber (HCPCF). Axially aligned air channels inside the HCPCF provide an excellent platform for optical sensing using SERS. In addition to the flexibility of optical fibers, HCPCF provides better light confinement and a larger interaction length for the guided light and the analyte, resulting in an improvement in sensitivity to detect low concentrations of bioanalytes in extremely low sample volumes. Herein, for the first time, we demonstrate the sensitive multiplex detection of biomarkers immobilized inside the HCPCF using antibody-conjugated SERS-active nanoparticles (SERS nanotags). As a proof-of-concept for targeted multiplex detection, initially we carried out the sensing of epidermal growth factor receptor (EGFR) biomarker in oral squamous carcinoma cell lysate using three different SERS nanotags. Subsequently, we also achieved simultaneous detection of hepatocellular carcinoma (HCC) biomarkers-alpha fetoprotein (AFP) and alpha-1-antitrypsin (A1AT) secreted in the supernatant from Hep3b cancer cell line. Using a SERS-HCPCF sensing platform, we could successfully demonstrate the multiplex detection in an extremely low sample volume of ∼20 nL. In future, this study may lead to sensitive biosensing platform for the low concentration detection of biomarkers in an extremely low sample volume of body fluids to achieve early diagnosis of multiple diseases. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). PMID:23963680

  14. Fulvic acid promotes extracellular anti-cancer mediators from RAW 264.7 cells, causing to cancer cell death in vitro.

    PubMed

    Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Dilshara, Matharage Gayani; Kang, Chang-Hee; Lee, Seungheon; Choi, Yung Hyun; Jeong, Yong Kee; Kim, Gi-Young

    2016-07-01

    Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor N(G)-monomethyl-l-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis. PMID:27177083

  15. miR-208-3p promotes hepatocellular carcinoma cell proliferation and invasion through regulating ARID2 expression

    SciTech Connect

    Yu, Peng; Wu, Dingguo; You, Yu; Sun, Jing; Lu, Lele; Tan, Jiaxing; Bie, Ping

    2015-08-15

    MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level. miRNA dysregulation plays a causal role in cancer progression. In this study, miR-208-3p was highly expressed and directly repressed ARID2 expression. As a result, ARID2 expression in hepatocellular carcinoma (HCC) was decreased. In vitro, miR-208-3p down-regulation and ARID2 over-expression elicited similar inhibitory effects on HCC cell proliferation and invasion. In vivo test results revealed that miR-208-3p down-regulation inhibited HCC tumorigenesis in Hep3B cells. Moreover, ARID2 was possibly a downstream element of transforming growth factor beta1 (TGFβ1)/miR-208-3p/ARID2 regulatory pathway. These findings suggested that miR-208-3p up-regulation is associated with HCC cell progression and may provide a new target for liver cancer treatment. - Highlights: • miR-208-3p was highly expressed and directly repressed the expression of ARID2 in HCC. • miR-208-3p contributed to HCC cell progression both in vitro and in vivo. • Over-expression of ARID2 inhibited the HCC cell proliferation and invasion. • Restoration of ARID2 partly reversed the the effect of miR-208-3p down-regulation on HCC cells. • Newly regulatory pathway: miR-208-3p mediated the repression of ARID2 by TGFβ1 in HCC cells.

  16. Expanded and Activated Natural Killer Cells for Immunotherapy of Hepatocellular Carcinoma.

    PubMed

    Kamiya, Takahiro; Chang, Yu-Hsiang; Campana, Dario

    2016-07-01

    Viral infection of the liver is a major risk factor for hepatocellular carcinoma (HCC). Natural killer (NK) cells recognize virally infected and oncogenically transformed cells, suggesting a therapeutic role for NK-cell infusions in HCC. Using the K562-mb15-41BBL cell line as a stimulus, we obtained large numbers of activated NK cells from the peripheral blood of healthy donors. Expanded NK cells exerted remarkably high cytotoxicity against HCC cell lines, which was generally much higher than that of unstimulated or IL2-activated NK cells. In immunodeficient NOD/scid IL2RGnull mice engrafted with Hep3B, treatment with expanded NK cells markedly reduced tumor growth and improved overall survival. HCC cells exposed for 48 hours to 5 μmol/L of sorafenib, a kinase inhibitor currently used for HCC treatment, remained highly sensitive to expanded NK cells. HCC cell reductions of 39.2% to 53.8% caused by sorafenib in three cell lines further increased to 80.5% to 87.6% after 4 hours of culture with NK cells at a 1:1 effector-to-target ratio. NK-cell cytotoxicity persisted even in the presence of sorafenib. We found that NKG2D, an NK-cell-activating receptor, was an important mediator of anti-HCC activity. We therefore enhanced its signaling capacity with a chimeric NKG2D-CD3ζ-DAP10 receptor. This considerably increased the anti-HCC cytotoxicity of expanded NK cells in vitro and in immunodeficient mice. The NK expansion and activation method applied in this study has been adapted to clinical-grade conditions. Hence, these results warrant clinical testing of expanded NK-cell infusions in patients with HCC, possibly after genetic modification with NKG2D-CD3ζ-DAP10. Cancer Immunol Res; 4(7); 574-81. ©2016 AACR. PMID:27197065

  17. Evaluation of Anticancer, Antioxidant, and Possible Anti-inflammatory Properties of Selected Medicinal Plants Used in Indian Traditional Medication.

    PubMed

    Shaikh, Rafik; Pund, Mahesh; Dawane, Ashwini; Iliyas, Sayyed

    2014-10-01

    The present study was carried out to evaluate the anticancer, antioxidant, and possible anti-inflammatory properties of diverse medicinal plants frequently used in Indian traditional medication. The selected botanicals such as Soymida fembrifuga (Roxb.) A. Juss. (Miliaceae), Tinospora cordifolia (Willd.) Miers. (Menispermaceae), Lavandula bipinnata (L.) O. Ktze. (Lamiaceae), and Helicteres isora L. (Sterculiaceae) extracted in different solvents were evaluated for their in vitro anticancer and antioxidant activities. The results obtained indicate that H. isora has potent cytotoxic activity toward the selected cancer cells such as HeLa-B75 (34.21 ± 0.24%), HL-60 (30.25 ± 1.36%), HEP-3B (25.36 ± 1.78%), and PN-15 (29.21 ± 0.52%). Interestingly, the selected botanicals selectively inhibited cyclooxygenase-2 (COX-2) more than (COX-1), which are the key enzymes implicated in inflammation. COX-2 inhibition was observed to be in the range of 19.66-49.52% as compared to COX-1 inhibition (3.93-19.61%). The results of the antioxidant study revealed that the selected plants were found to be effective 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH), and superoxide radical (SOR) scavenging agents. High-performance thin layer chromatography (HPTLC) fingerprint of flavonoids was used as a measure of quality control of the selected plant samples. The results of the present findings strengthen the potential of the selected plants as a resource for the discovery of novel anticancer, anti-inflammatory, and antioxidant agents. PMID:25379467

  18. Ganetespib radiosensitization for liver cancer therapy

    PubMed Central

    Chettiar, Sivarajan T.; Malek, Reem; Annadanam, Anvesh; Nugent, Katriana M.; Kato, Yoshinori; Wang, Hailun; Cades, Jessica A.; Taparra, Kekoa; Belcaid, Zineb; Ballew, Matthew; Manmiller, Sarah; Proia, David; Lim, Michael; Anders, Robert A.; Herman, Joseph M.; Tran, Phuoc T.

    2016-01-01

    ABSTRACT Therapies for liver cancer particularly those including radiation are still inadequate. Inhibiting the stress response machinery is an appealing anti-cancer and radiosensitizing therapeutic strategy. Heat-shock-protein-90 (HSP90) is a molecular chaperone that is a prominent effector of the stress response machinery and is overexpressed in liver cancer cells. HSP90 client proteins include critical components of pathways implicated in liver cancer cell survival and radioresistance. The effects of a novel non-geldanamycin HSP90 inhibitor, ganetespib, combined with radiation were examined on 3 liver cancer cell lines, Hep3b, HepG2 and HUH7, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γH2AX foci kinetics and client protein expression in pathways important for liver cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined ganetespib-radiation treatment on tumor cell proliferation in a HepG2 hind-flank tumor graft model. Nanomolar levels of ganetespib alone exhibited liver cancer cell anti-cancer activity in vitro as shown by decreased clonogenic survival that was associated with increased apoptotic cell death, prominent G2-M arrest and marked changes in PI3K/AKT/mTOR and RAS/MAPK client protein activity. Ganetespib caused a supra-additive radiosensitization in all liver cancer cell lines at low nanomolar doses with enhancement ratios between 1.33–1.78. These results were confirmed in vivo, where the ganetespib-radiation combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in HepG2 tumor grafts. Our data suggest that combined ganetespib-radiation therapy exhibits promising activity against liver cancer cells, which should be investigated in clinical studies. PMID:26980196

  19. The ruthenium compound KP1339 potentiates the anticancer activity of sorafenib in vitro and in vivo☆

    PubMed Central

    Heffeter, Petra; Atil, Bihter; Kryeziu, Kushtrim; Groza, Diana; Koellensperger, Gunda; Körner, Wilfried; Jungwirth, Ute; Mohr, Thomas; Keppler, Bernhard K.; Berger, Walter

    2013-01-01

    KP1339 is a promising ruthenium-based anticancer compound in early clinical development. This study aimed to test the effects of KP1339 on the in vitro and in vivo activity of the multi-kinase inhibitor sorafenib, the current standard first-line therapy for advanced hepatoma. Anticancer activity of the parental compounds as compared to the drug combination was tested against a panel of cancer cell lines with a focus on hepatoma. Combination of KP1339 with sorafenib induced in the majority of all cases distinctly synergistic effects, comprising both sorafenib-resistant as well as sorafenib-responsive cell models. Several mechanisms were found to underlie these multifaceted synergistic activities. Firstly, co-exposure induced significantly enhanced accumulation levels of both drugs resulting in enhanced apoptosis induction. Secondly, sorafenib blocked KP1339-mediated activation of P38 signalling representing a protective response against the ruthenium drug. In addition, sorafenib treatment also abrogated KP1339-induced G2/M arrest but resulted in check point-independent DNA-synthesis block and a complete loss of the mitotic cell populations. The activity of the KP1339/sorafenib combination was evaluated in the Hep3B hepatoma xenograft. KP1339 monotherapy led to a 2.4-fold increase in life span and, thus, was superior to sorafenib, which induced a 1.9-fold prolonged survival. The combined therapy further enhanced the mean survival by 3.9-fold. Synergistic activity was also observed in the VM-1 melanoma xenograft harbouring an activating braf mutation. Together, our data indicate that the combination of KP1339 with sorafenib displays promising activity in vitro and in vivo especially against human hepatoma models. PMID:23790465

  20. Lactobionic acid-modified dendrimer-entrapped gold nanoparticles for targeted computed tomography imaging of human hepatocellular carcinoma.

    PubMed

    Liu, Hui; Wang, Han; Xu, Yanhong; Guo, Rui; Wen, Shihui; Huang, Yunpeng; Liu, Weina; Shen, Mingwu; Zhao, Jinglong; Zhang, Guixiang; Shi, Xiangyang

    2014-05-14

    Development of novel nanomaterial-based contrast agents for targeted computed tomography (CT) imaging of tumors still remains a great challenge. Here we describe a novel approach to fabricating lactobionic acid (LA)-modified dendrimer-entrapped gold nanoparticles (LA-Au DENPs) for in vitro and in vivo targeted CT imaging of human hepatocellular carcinoma. In this study, amine-terminated poly(amidoamine) dendrimers of generation 5 pre-modified with fluorescein isothiocyanate and poly(ethylene glycol)-linked LA were employed as templates to form Au nanoparticles. The remaining dendrimer terminal amines were subjected to an acetylation reaction to form LA-Au DENPs. The prepared LA-Au DENPs were characterized via different methods. Our results reveal that the multifunctional Au DENPs with a Au core size of 2.7 nm have good stability under different pH (5-8) and temperature (4-50 °C) conditions and in different aqueous media, and are noncytotoxic to normal cells but cytotoxic to the targeted hepatocarcinoma cells in the given concentration range. In vitro flow cytometry data show that the LA-Au DENPs can be specifically uptaken by a model hepatocarcinoma cell line overexpressing asialoglycoprotein receptors through an active receptor-mediated targeting pathway. Importantly, the LA-Au DENPs can be used as a highly effective nanoprobe for specific CT imaging of hepatocarcinoma cells in vitro and the xenoplanted tumor model in vivo. The developed LA-Au DENPs with X-ray attenuation property greater than clinically employed iodine-based CT contrast agents hold a great promise to be used as a nanoprobe for targeted CT imaging of human hepatocellular carcinoma. PMID:24712914

  1. Photodynamic activity of BAM-SiPc, an unsymmetrical bisamino silicon(IV) phthalocyanine, in tumour-bearing nude mice

    PubMed Central

    Leung, S C H; Lo, P-C; Ng, D K P; Liu, W-K; Fung, K-P; Fong, W-P

    2008-01-01

    Background and purpose Ever since the discovery of photodynamic therapy, there has been a continuous search for more potent photosensitizers. Towards that end, we have synthesized a number of novel phthalocyanine derivatives. The unsymmetrical bisamino silicon(IV) phthalocyanine BAM-SiPc is one of the most potent compounds. In in vitro cell culture, it exhibits high phototoxicity against a number of cancer cell lines. Experimental approach In the present investigation, the in vivo effect of BAM-SiPc was studied in the tumour-bearing nude mice model. The biodistribution of BAM-SiPc was followed to evaluate its tumour selectivity and rate of clearance. The tumour volume in the hepatocarcinoma HepG2- and the colorectal adenocarcinoma HT29-bearing nude mice was measured after photodynamic therapy. The level of intrinsic toxicity induced was also investigated. Finally, the metabolism of BAM-SiPc in the ‘normal' WRL68 liver cells and the hepatocarcinoma HepG2 cells was compared. Key results The results not only showed significant tumour regression of HepG2 and growth inhibition of HT29 in the tumour-bearing nude mice, but also no apparent hepatic or cardiac injury with the protocol used. Histological analyses showed that apoptosis was induced in the solid tumour. BAM-SiPc could be metabolized by WRL68 liver cells but not by the hepatocarcinoma HepG2 cells. Unfortunately, BAM-SiPc did not show any specific targeting towards the tumour tissue. Conclusions and implications The efficiency of BAM-SiPc in inhibiting tumour growth makes it a good candidate for further evaluation. Enhancement of its uptake in tumour tissue by conjugation with biomolecules is currently under investigation. PMID:18332853

  2. The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

    SciTech Connect

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

    2010-07-16

    Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

  3. Mirando al Futuro del Desarrollo Humano en America Latina y el Caribe. Seminario Regional sobre Universalization de la Educacion (Sucre, Bolivia, 4-10 mayo 1987) (Looking at the Future of Human Development in Latin America and the Caribbean. Regional Seminar on the Universalization of Education [Sucre, Bolivia, May 4-10, 1987]).

    ERIC Educational Resources Information Center

    United Nations Children's Fund, Santiago (Chile). Oficina Regional para les Americas.

    One of every two children in developing nations does not complete primary school and four out of ten adults do not read or write. Of these ten, six are women. There were 44 million illiterate adults in Latin America in 1985, and of the 66 million school age children, 8.5 million were not in school. Thirty million of these children lived in…

  4. Current evidence for the hepatoprotective activities of the medicinal mushroom Antrodia cinnamomea

    PubMed Central

    2013-01-01

    Antrodia cinnamomea (AC) is an endemic mushroom species of Taiwan, and has been demonstrated to possess diverse biological and pharmacological activities, such as anti-hypertension, anti-hyperlipidemia, anti-inflammation, anti-oxidation, anti-tumor, and immunomodulation. This review focuses on the inhibitory effects of AC on hepatitis, hepatocarcinoma, and alcohol-induced liver diseases (e.g., fatty liver, fibrosis). The relevant biochemical and molecular mechanisms are addressed. Overall, this review summarizes the hepatoprotective activities in vitro and in vivo. However, there is no doubt that human and clinical trials are still limited, and further studies are required for the development of AC-related products. PMID:24180549

  5. Stimulation of anti-tumour immunity in guinea-pigs by methanol extraction residue of BCG.

    PubMed Central

    Wainberg, M. A.; Deutsch, V.; Weiss, D. W.

    1976-01-01

    The immunoprophylactic effects of the methanol extraction residue (MER) of BCG were investigated in Strain 2 guinea-pigs injected with cells of the transplantable, diethylnitrosamine-induced, Line 10 hepatocarcinoma. Pretreatment with MER at times ranging from 18 to 182 days prior to tumour implantation protected approximately 40% of guinea-pigs from progressive neoplastic disease. In addition, MER-treated animals developed specific cell-mediated anti-tumour immunity both more rapidly and at higher levels than did non-MER-treated tumour-bearing controls. It was not possible, however, to prognosticate from the results of such laboratory studies to the outcome of immunoprophylaxis. PMID:187207

  6. Immunotherapy of Metastases Enhances Subsequent Chemotherapy

    NASA Astrophysics Data System (ADS)

    Hanna, Michael G.; Key, Marc E.

    1982-07-01

    In many multimodal therapies of cancer, postsurgical chemotherapy is administered before immunotherapy for treatment of micrometastatic disease. This sequence may not be the most efficacious. Experiments in which strain 2 guinea pigs bearing syngeneic L10 hepatocarcinomas were given immunotherapy showed that infiltrating immune effector cells not only were tumoricidal but disrupted the characteristically compact structure of metastatic foci. When cytotoxic drugs were administered at the peak of this inflammatory response, the survival rate of the guinea pigs increased significantly. We conclude that postsurgical immunotherapy can enhance the effect of cytotoxic drugs administered subsequently.

  7. Dietary Factors and Hepatoma in Rainbow Trout (Salmo gairdneri). I. Aflatoxins in Vegetable Protein Feedstuffs

    USGS Publications Warehouse

    Sinnhuber, R.O.; Wales, J.H.; Ayers, J.L.; Engebrecht, R.H.; Amend, D.F.

    1968-01-01

    Aflatoxins (toxic metabolites of the mold Aspergillus flavus) were present in a commercial trout ration causing hepatoma in rainbow trout. Cottonseed meal and solvent extracts of cottonseed meal and of rations containing cottonseed meal and peanut meal were found by chemical assay and confirmed by duckling assay to contain aflatoxins. Diets containing these materials and a purified test diet to which aflatoxins had been added produced microscopic tumors in 6 months and gross lesions of hepatocarcinoma in 9 months. Similar diets without aflatoxin were negative.

  8. Synchronous Fibrolamellar Hepatocellular Carcinoma and Auricular Myxoma

    PubMed Central

    González-Cantú, Yessica M.; Rodriguez-Padilla, Cristina; Tena-Suck, Martha Lilia; García de la Fuente, Alberto; Mejía-Bañuelos, Rosa María; Díaz Mendoza, Raymundo; Quintanilla-Garza, Samuel; Batisda-Acuña, Yolaester

    2015-01-01

    Synchronic occurrence of benign and malignant tumors is extremely rare. Fibrolamellar hepatocellular carcinoma represents 1% to 2% of all hepatocarcinomas, while myxomas represent about half of all the cases of primary tumors of the heart. We present the case of a 53-year-old woman with a left atrial myxoma that was surgically removed. Several weeks later, the patient returned to the hospital with abdominal pain. CT scan showed a mass in the left lobe of the liver that was resected and diagnosed as fibrolamellar hepatocellular carcinoma. As of this writing, the patient is healthy. PMID:26509093

  9. Targeting the inflammation in HCV-associated hepatocellular carcinoma: a role in the prevention and treatment

    PubMed Central

    2010-01-01

    Epidemiological, preclinical and clinical studies demonstrated that chronic inflammation induced by hepatitis C virus (HCV) is crucial in hepatocellular carcinogenesis. The interaction between hepatocytes and microenvironment regards virus, inflammatory and immunocompetent cells, chemo- and cyto-kines, reactive oxygen species (ROS) and nitric oxide (NO), generating cell transformation. We suggest hepatocarcinoma (HCC) as a model in which the targeting of microenvironment determine neoplastic transformation. The present review focuses on: the role of inflammation in carcinogenesis, the clinical impact of HCC and the inadequacy of the actual therapy, the chemoprevention targeting the microenvironment. PMID:21047421

  10. Synchronous Fibrolamellar Hepatocellular Carcinoma and Auricular Myxoma.

    PubMed

    González-Cantú, Yessica M; Rodriguez-Padilla, Cristina; Tena-Suck, Martha Lilia; García de la Fuente, Alberto; Mejía-Bañuelos, Rosa María; Díaz Mendoza, Raymundo; Quintanilla-Garza, Samuel; Batisda-Acuña, Yolaester

    2015-01-01

    Synchronic occurrence of benign and malignant tumors is extremely rare. Fibrolamellar hepatocellular carcinoma represents 1% to 2% of all hepatocarcinomas, while myxomas represent about half of all the cases of primary tumors of the heart. We present the case of a 53-year-old woman with a left atrial myxoma that was surgically removed. Several weeks later, the patient returned to the hospital with abdominal pain. CT scan showed a mass in the left lobe of the liver that was resected and diagnosed as fibrolamellar hepatocellular carcinoma. As of this writing, the patient is healthy. PMID:26509093

  11. Dynamic interaction of /sup 111/indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy

    SciTech Connect

    Hwang, K.M.; Keenan, A.M.; Frincke, J.; David, G.; Pearson, J.; Oldham, R.K.; Morgan, A.C. Jr.

    1986-05-01

    Two /sup 111/indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.

  12. Potentiation of antiproliferative drug activity by lonidamine in hepatocellular carcinoma cells.

    PubMed

    Ricotti, L; Tesei, A; De Paola, F; Milandri, C; Amadori, D; Frassineti, G L; Ulivi, P; Zoli, W

    2003-10-01

    The ability of lonidamine (LND), a derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of anticancer drugs was investigated in two human hepatocarcinoma (HCC) cell lines. The cytotoxicity of drugs used singly, in association or in sequence was evaluated using the Sulforhodamine B (SRB) assay. LND did not appreciably potentiate the effect of antitumor drugs when given before or simultaneously, in either cell line. Conversely, a synergistic interaction was observed in both cell lines when LND was given after conventional drugs. LND produced a moderate decrease in S-phase cell fraction and did not induce apoptosis. Conversely, paclitaxel (TAX) induced an important block in G2 and an increase in apoptosis. Following a 48-h TAX wash out, a progressive passage of cells from G2 to M phase was observed with a corresponding increase in apoptotic cells. Post-treatment with LND increased the cytotoxicity of some antitumor drugs, especially TAX, in hepatocarcinoma cells, possibly by preventing, as an energolytic drug, cell damage repair or by producing an additional effect on microtubule stabilization. PMID:14598941

  13. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

    PubMed Central

    Lee, Kuan-Han; Ho, Wen-Yueh; Wu, Shu-Jing; Omar, Hany A.; Huang, Po-Jui; Wang, Clay C. C.; Hung, Jui-Hsiang

    2014-01-01

    Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy. PMID:24406729

  14. Inhibitory effect of midkine-binding peptide on tumor proliferation and migration

    PubMed Central

    Huang, Hui-Lian; Shen, Jian-Fen; Min, Li-Shan; Ping, Jin-Liang; Lu, Yong-Liang; Dai, Li-Cheng

    2015-01-01

    Background: To investigate the inhibitory effect of midkine-binding peptides on human umbilical vein endothelial cells (HUVECs) proliferation and angiogenesis of xenograft tumor. Methods: The midkine-binding peptides were panned by Ph.D.-7™ Phage Display Peptide Library Kit, and the specific binding activities of positive clones to target protein were examined by phage ELISA. The effect of midkine-binding peptides on proliferation of HUVECs was confirmed by MTT test. The xenograft tumor model was formed in BALB/c mice with the murine hepatocarcinoma cells H22 (H22). Microvessel density (MVD) was analyzed by immunohistochemistry of factor VIII staining. Results: Midkine-binding peptides have the inhibitory effects on tumor angiogenesis, a proliferation assay using human umbilical vein endothelial cells (HUVECs) indicated that particular midkine-binding peptides significantly inhibited the proliferation of the HUVECs. Midkine-binding peptides were also observed to efficiently suppress angiogenesis induced by murine hepatocarcinoma H22 cells in BALB/c nude mice. Conclusion: The midkine-binding peptides can inhibit solid tumor growth by retarding the formation of new blood vessels. The results indicate that midkine-binding peptides may represent potent anti-angiogenesis agents in vivo. PMID:26191241

  15. The analysis of N-glycolylneuraminic acid(NeuGc) of hepatoma tissue and K562 cell ferritins using HPLC and mass spectrometry.

    PubMed

    Asakawa, Hideo; Sasabe, Masataka; Miyazaki, Ryuunosuke; Matsuda, Haruo; Fukai, Fumio; Hanada, Kazuki; Hirano, Hisashi; Takasaki, Seiichi

    2006-07-01

    Ferritin is an iron-storage protein and its serum level is known to increase in the patient of with inflammation and malignant tumor. To further elucidate the difference between ferritins from normal human liver tissue and that of cancer cells, their sialic acids were analyzed. The Western blot analysis and the cytochemical staining using anti-NeuGc antiserum indicated that ferritins from the human hepatocarcinoma tissue and malignant K562 cells contain NeuGc, but that from the normal liver does not. The result was also confirmed by HPLC analysis and MALDI-TOF/MS analysis of sialic acids which were derivatized by the DMB method. It was also shown that the sialic acid content in hepatocarcinoma ferritin was much higher than that in the normal liver ferritin. These results suggest that normal and cancerous liver ferritins are qualitatively and quantitatively different in sialylation. In addition, K562 cells were shown to express NeuGc even if the cells were cultured in serum-free media which lack NeuGc. This is of interest from the current concept that expression of NeuGc in human cells is due to uptake and utilization of exogenous NeuGc. PMID:25792781

  16. Screening and identification of a specific peptide for targeting hypoxic hepatoma cells.

    PubMed

    Liu, Yiming; Xia, Xiangwen; Wang, Yong; Li, Xin; Zhou, Guofeng; Liang, Huiming; Feng, Gansheng; Zheng, Chuansheng

    2016-08-01

    The biological behaviors of residual hepatoma cells after transarterial embolization therapy, which exist in a hypoxic or even anaerobic tumor microenvironment, differ from the tumor cells under normoxic conditions. This study aimed to use a phage display peptide library for in vivo and in vitro screening to obtain a peptide which could specifically bind to hypoxic hepatoma cells, allowing further targeted diagnosis and treatment for liver cancer. In this study, hypoxic hepatoma cells HepG2 (targeted cells), and normal liver cells HL-7702 (control cells), were utilized to perform three rounds of in vitro screening using a phage-displayed 7-mer peptide library. In addition, hypoxic HepG2 were subcutaneously injected into nude mice to establish a hepatocarcinoma model, followed by performing three rounds of in vivo screening on the phages identified from the in vitro screening. The products from the screening were further identified using ELISA and immunofluorescence staining on cells and tissues. The results indicated that the P11 positive clone had the highest binding effect with hypoxic hepatoma cells. The sequence of the exogenous insert fragment of P11 positive clone was obtained by sequencing: GSTSFSK. The binding assay indicated that GSTSFSK could specifically bind to hypoxic hepatoma cells and hepatocarcinoma tissues. This 7-mer peptide has the potential to be developed as an useful molecular to the targeting diagnosis and treatment of residual hepatoma cells after transarterial chemoembolization. PMID:27381416

  17. Creatine kinase activity and isoenzymes in lung, colon and liver carcinomas.

    PubMed Central

    Joseph, J.; Cardesa, A.; Carreras, J.

    1997-01-01

    We have compared the levels of creatine kinase (CK) activity and the distribution of CK isoenzymes determined by agarose gel electrophoresis in normal colon, liver and lung tissues, and in colon, liver and lung adenocarcinomas, lung squamous cell carcinomas and lung carcinoids. Colon and lung adenocarcinomas, and squamous cell carcinomas presented lower CK activity than the normal tissues and no differences were found between hepatocarcinoma and normal liver tissue. In contrast, lung carcinoids had higher CK activity than normal lung tissue. Type BB-CK was the predominant isoenzyme in normal lung, colon and liver tissues. Type MM isoenzyme was detected in normal lung and type MB-CK was found in normal colon. In most lung tumours the CK isoenzyme electrophoretic pattern did not change. However, no type BB-CK was detected in some hepatocarcinomas, type MM-CK decreased in lung carcinoids and type MB isoenzyme was not observed in colon adenocarcinomas. It is concluded that in most tumours there is a decrease in the expression of type B- and type M-CK subunits, whereas in lung carcinoid the expression of type B-CK activity increases. Thus, the increase in type BB-CK observed in the serum of patients with lung and colon adenocarcinomas is probably due mainly to enhanced enzyme release as a result of tumour cell necrosis. Images Figure 1 Figure 2 Figure 3 PMID:9303358

  18. Natural History of Helicobacter hepaticus Infection in Conventional A/J Mice, with Special Reference to Liver Involvement

    PubMed Central

    Avenaud, Philippe; Le Bail, Brigitte; Mayo, Kathryn; Marais, Armelle; Fawaz, Rabia; Bioulac-Sage, Paulette; Megraud, Francis

    2003-01-01

    It has been reported that Helicobacter hepaticus infection of mice leads to chronic hepatitis and hepatocarcinoma. Our aim was to monitor a cohort of 80 conventional A/J mice in which half of the mice were infected by H. hepaticus in order to study the evolution of the infection and the pathological changes in comparison to uninfected mice. H. hepaticus was detected by culture only in some colon and cecum specimens after 17 months of age, while PCR detected H. hepaticus in the intestines of all inoculated mice after only 5 months of infection. The percentage of mice in which H. hepaticus was detected in the gallbladder, bile ducts, and liver by PCR, as well as the number of bacteria present in the liver, tended to increase with increasing age and longer infection time. Anti-H. hepaticus immunoglobulin G antibodies were positive by enzyme-linked immunosorbent assay only in inoculated mice. Pathological findings were also more frequent as the mice grew older: fibrosis was present (especially in the peripheral part of the liver), and significant portal inflammation including lymphoid nodules was present in almost all infected animals. Biliary lesions of neutrophilic acute cholangitis or lymphocytic cholangitis were noted. However, lesions were also observed in uninfected animals, although at a significantly lower level, and the only hepatocellular carcinoma occurred in an uninfected mouse. The evolution towards hepatocarcinoma is not always the endpoint and may depend on the bacterial strain and on the environmental conditions. PMID:12761159

  19. Grados de comprobación de estudios de terapias integrales (PDQ®)—Versión para profesionales de salud

    Cancer.gov

    Información acerca de cómo sopesar la fuerza de los datos probatorios obtenidos de los estudios de investigación sobre terapias integrales, alternativas y complementarias en seres humanos con cáncer.

  20. Azathioprine desensitizes liver cancer cells to insulin-like growth factor 1 and causes apoptosis when it is combined with bafilomycin A1

    SciTech Connect

    Hernández-Breijo, Borja; Monserrat, Jorge; Román, Irene D.; González-Rodríguez, Águeda; Fernández-Moreno, M. Dolores; and others

    2013-11-01

    Hepatoblastoma is a primary liver cancer that affects children, due to the sensitivity of this tumor to insulin-like growth factor 1 (IGF-1). In this paper we show that azathioprine (AZA) is capable of inhibiting IGF1-mediated signaling cascade in HepG2 cells. The efficiency of AZA on inhibition of proliferation differs in the evaluated cell lines as follows: HepG2 (an experimental model of hepatoblastoma) > Hep3B (derived from a hepatocellular carcinoma) > HuH6 (derived from a hepatoblastoma) ≫ HuH7 (derived from a hepatocellular carcinoma) = Chang Liver cells (a non-malignant cellular model). The effect of AZA in HepG2 cells has been proven to derive from activation of Ras/ERK/TSC2, leading to activation of mTOR/p70S6K in a sustained manner. p70S6K phosphorylates IRS-1 in serine 307 which leads to the uncoupling between IRS-1 and p85 (the regulatory subunit of PI3K) and therefore causing the lack of response of HepG2 to IGF-1. As a consequence, proliferation induced by IGF-1 is inhibited by AZA and autophagy increases leading to senescence of HepG2 cells. Our results suggest that AZA induces the autophagic process in HepG2 activating senescence, and driving to deceleration of cell cycle but not to apoptosis. However, when simultaneous to AZA treatment the autophagy was inhibited by bafilomycin A1 and the degradation of regulatory proteins of cell cycle (e.g. Rb, E2F, and cyclin D1) provoked apoptosis. In conclusion, AZA induces resistance in hepatoblastoma cells to IGF-1, which leads to autophagy activation, and causes apoptosis when it is combined with bafilomycin A1. We are presenting here a novel mechanism of action of azathioprine, which could be useful in treatment of IGF-1 dependent tumors, especially in its combination with other drugs. - Highlights: • Azathioprine activated Ras/ERK/TSC-2/mTOR/p70S6K signaling pathway in HepG2 cells. • Azathioprine inhibited IGF-1-mediated signaling cascade. • Azathioprine induced autophagy leading to cell cycle

  1. Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response.

    PubMed

    Kim, Min Sun; Shigenaga, Judy; Moser, Art; Grunfeld, Carl; Feingold, Kenneth R

    2004-10-01

    The acute-phase response (APR) induces alterations in lipid metabolism, and our data suggest that this is associated with suppression of type II nuclear hormone receptors that are key regulators of fatty acid, cholesterol, and bile acid metabolism. Recently, the farnesoid X receptor (FXR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) were found to regulate DHEA sulfotransferase (Sult2A1), which plays an important role in DHEA sulfation and detoxification of bile acids. Because FXR, PXR, and CAR are suppressed during the APR, we hypothesized that Sult2A1 is downregulated during the APR. To induce the APR, mice were treated with LPS, which will then trigger the release of various cytokines, and the mRNA levels of Sult2A1 and the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2), as well as the enzyme activity of Sult2A1, were determined in the liver. We found that mRNA levels of Sult2A1 decrease in a time- and dose-dependent manner during the LPS-induced APR. Similar changes were observed in the mRNA levels of PAPSS2, the major synthase of PAPS in the liver. Moreover, hepatic Sult2A1 activity and serum levels of DHEA-sulfate (DHEA-S) were significantly decreased in LPS-treated animals. These results suggest that decreased levels or activities of FXR, PXR, and CAR during the APR could contribute to decreases in Sult2A1, resulting in decreased sulfation of DHEA and lower circulating level of DHEA-S. Finally, we found that both TNF and IL-1 caused a significant decrease in the mRNA level of Sult2A1 in Hep3B human hepatoma cells, suggesting that the proinflammatory cytokines TNF and IL-1 mediate the inhibitory effect of LPS on Sult2A1 mRNA level. Our study provides a possible mechanism by which infection and inflammation are associated with altered steroid metabolism and cholestasis. PMID:15198932

  2. Molecular modelling, synthesis, cytotoxicity and anti-tumour mechanisms of 2-aryl-6-substituted quinazolinones as dual-targeted anti-cancer agents

    PubMed Central

    Hour, M J; Lee, K H; Chen, T L; Lee, K T; Zhao, Yu; Lee, H Z

    2013-01-01

    Background and Purpose Our previous study demonstrated that 6-(pyrrolidin-1-yl)-2-(3-methoxyphenyl)quinazolin-4-one (HMJ38) was a potent anti-tubulin agent. Here, HMJ38 was used as a lead compound to develop more potent anti-cancer agents and to examine the anti-cancer mechanisms. Experimental Approach Using computer-aided drug design, 2-aryl-6-substituted quinazolinones (MJ compounds) were designed and synthesized by introducing substituents at C-2 and C-6 positions of HMJ38. The cytotoxicity of MJ compounds towards human cancer cells was examined by Trypan blue exclusion assay. Microtubule distribution was visualized using TubulinTracker™ Green reagent. Protein expression of cell cycle regulators and JNK was assessed by Western blot analysis. Key Results Compounds MJ65–70 exhibited strong anti-proliferative effects towards melanoma M21, lung squamous carcinoma CH27, lung non-small carcinoma H460, hepatoma Hep3B and oral cancer HSC-3 cells, with one compund MJ66 (6-(pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one) highly active against M21 cells (IC50 about 0.033 μM). Treatment of CH27 or HSC-3 cells with MJ65–70 resulted in significant mitotic arrest accompanied by increasing multiple asters of microtubules. JNK protein expression was involved in the MJ65–70-induced CH27 and M21 cell death. Consistent with the cell cycle arrest at G2/M phase, marked increases in cyclin B1 and Bcl-2 phosphorylation were also observed, after treatment with MJ65–70. Conclusions and Implication MJ65–70 are dual-targeted, tubulin- and JNK-binding, anti-cancer agents and induce cancer cell death through up-regulation of JNK and interfering in the dynamics of tubulin. Our work provides a new strategy and mechanism for developing dual-targeted anti-cancer drugs, contributing to clinical anti-cancer drug discovery and application. PMID:23638624

  3. The Relative Biological Effectiveness for Carbon and Oxygen Ion Beams Using the Raster-Scanning Technique in Hepatocellular Carcinoma Cell Lines

    PubMed Central

    Habermehl, Daniel; Ilicic, Katarina; Dehne, Sarah; Rieken, Stefan; Orschiedt, Lena; Brons, Stephan; Haberer, Thomas; Weber, Klaus-Josef; Debus, Jürgen; Combs, Stephanie E.

    2014-01-01

    Background Aim of this study was to evaluate the relative biological effectiveness (RBE) of carbon (12C) and oxygen ion (16O)-irradiation applied in the raster-scanning technique at the Heidelberg Ion beam Therapy center (HIT) based on clonogenic survival in hepatocellular carcinoma cell lines compared to photon irradiation. Methods Four human HCC lines Hep3B, PLC, HepG2 and HUH7 were irradiated with photons, 12C and 16O using a customized experimental setting at HIT for in-vitro trials. Cells were irradiated with increasing physical photon single doses of 0, 2, 4 and 6 Gy and heavy ionsingle doses of 0, 0.125, 0.5, 1, 2, 3 Gy (12C and 16O). SOBP-penetration depth and extension was 35 mm +/−4 mm and 36 mm +/−5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for 12C, and 154 MeV/u and 146 keV/um for 16O. Clonogenic survival was computated and realtive biological effectiveness (RBE) values were defined. Results For all cell lines and both particle modalities α- and β-values were determined. As expected, α-values were significantly higher for 12C and 16O than for photons, reflecting a steeper decline of the initial slope of the survival curves for high-LET beams. RBE-values were in the range of 2.1–3.3 and 1.9–3.1 for 12C and 16O, respectively. Conclusion Both irradiation with 12C and 16O using the rasterscanning technique leads to an enhanced RBE in HCC cell lines. No relevant differences between achieved RBE-values for 12C and 16O were found. Results of this work will further influence biological-adapted treatment planning for HCC patients that will undergo particle therapy with 12C or 16O. PMID:25460352

  4. Expression of hypoxia-inducible factor 3α in hepatocellular carcinoma and its association with other hypoxia-inducible factors

    PubMed Central

    LIU, PING; FANG, XIEFAN; SONG, YANG; JIANG, JIAN-XIN; HE, QIAN-JIN; LIU, XIANG-JIE

    2016-01-01

    The functional role of hypoxia-inducible factor (HIF)-3α in the development of hepatocellular carcinoma (HCC) is not yet fully understood. The aim of the present study was to elucidate the association between HIF-3α expression and the clinicopathological features as well as prognosis of HCC patients. In addition, we investigated the association between HIF-3α expression and the expression of HIF-1α and HIF-2α in tumor tissues. The protein levels of HIF-3α were determined using immunohistochemical analysis of paraffin sections of 126 paired HCC and peritumoral tissues. PLC/PRF/5 cells, a human HCC cell line, were transfected with HIF-1α and HIF-2α vectors and HIF-3α mRNA and protein expression was detected using quantitative polymerase chain reaction and western blot analysis, respectively. The expression of HIF-3α was upregulated in 46.0% (58/126) and downregulated in 42.9% (54/126) of tumor tissues, respectively, when compared to peritumoral tissues. HIF-3α protein expression was not associated with peripheral blood vessel invasion, overall survival, or disease-free survival in HCC patients (P>0.05). In HCC tissues, the levels of HIF-3α protein were positively correlated with HIF-2α, but not with HIF-1α expression in HCC tissues. HIF-3α was upregulated in PLC/PRF/5 and Hep3B cells overexpressed with HIF-1α or HIF-2α. The hypoxic microenvironment of liver cancer did not lead to elevated HIF-3α protein expression, indicating that HIF-3α is regulated differently from HIF-1α in vivo. The correlation between HIF-3α and HIF-2α expression at the cellular and tissue levels indicated that HIF-3α may be a target gene of HIF-2α. The hypoxic microenvironment did not lead to elevation of HIF-3α protein expression in liver cancer; thus, HIF-3α may be a target gene of HIF-2α. PMID:27284334

  5. Inflammation Promotes Expression of Stemness-Related Properties in HBV-Related Hepatocellular Carcinoma.

    PubMed

    Chang, Te-Sheng; Chen, Chi-Long; Wu, Yu-Chih; Liu, Jun-Jen; Kuo, Yung Che; Lee, Kam-Fai; Lin, Sin-Yi; Lin, Sey-En; Tung, Shui-Yi; Kuo, Liang-Mou; Tsai, Ying-Huang; Huang, Yen-Hua

    2016-01-01

    The expression of cancer stemness is believed to reduce the efficacy of current therapies against hepatocellular carcinoma (HCC). Understanding of the stemness-regulating signaling pathways incurred by a specific etiology can facilitate the development of novel targets for individualized therapy against HCC. Niche environments, such as virus-induced inflammation, may play a crucial role. However, the mechanisms linking inflammation and stemness expression in HCC remain unclear. Here we demonstrated the distinct role of inflammatory mediators in expressions of stemness-related properties involving the pluripotent octamer-binding transcription factor 4 (OCT4) in cell migration and drug resistance of hepatitis B virus-related HCC (HBV-HCC). We observed positive immunorecognition for macrophage chemoattractant protein 1 (MCP-1)/CD68 and OCT4/NANOG in HBV-HCC tissues. The inflammation-conditioned medium (inflamed-CM) generated by lipopolysaccharide-stimulated U937 human leukemia cells significantly increased the mRNA and protein levels of OCT4/NANOG preferentially in HBV-active (HBV+HBsAg+) HCC cells. The inflamed-CM also increased the side population (SP) cell percentage, green fluorescent protein (GFP)-positive cell population, and luciferase activity of OCT4 promoter-GFP/luciferase in HBV-active HCC cells. Furthermore, the inflamed-CM upregulated the expressions of insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) and activated IGF-IR/Akt signaling in HBV-HCC. The IGF-IR phosphorylation inhibitor picropodophyllin (PPP) suppressed inflamed-CM-induced OCT4 and NANOG levels in HBV+HBsAg+ Hep3B cells. Forced expression of OCT4 significantly increased the secondary sphere formation and cell migration, and reduced susceptibility of HBV-HCC cells to cisplatin, bleomycin, and doxorubicin. Taking together, our results show that niche inflammatory mediators play critical roles in inducing the expression of stemness-related properties involving IGF-IR activation, and

  6. Inflammation Promotes Expression of Stemness-Related Properties in HBV-Related Hepatocellular Carcinoma

    PubMed Central

    Wu, Yu-Chih; Liu, Jun-Jen; Kuo, Yung Che; Lee, Kam-Fai; Lin, Sin-Yi; Lin, Sey-En; Tung, Shui-Yi; Kuo, Liang-Mou; Tsai, Ying-Huang; Huang, Yen-Hua

    2016-01-01

    The expression of cancer stemness is believed to reduce the efficacy of current therapies against hepatocellular carcinoma (HCC). Understanding of the stemness-regulating signaling pathways incurred by a specific etiology can facilitate the development of novel targets for individualized therapy against HCC. Niche environments, such as virus-induced inflammation, may play a crucial role. However, the mechanisms linking inflammation and stemness expression in HCC remain unclear. Here we demonstrated the distinct role of inflammatory mediators in expressions of stemness-related properties involving the pluripotent octamer-binding transcription factor 4 (OCT4) in cell migration and drug resistance of hepatitis B virus-related HCC (HBV-HCC). We observed positive immunorecognition for macrophage chemoattractant protein 1 (MCP-1)/CD68 and OCT4/NANOG in HBV-HCC tissues. The inflammation-conditioned medium (inflamed-CM) generated by lipopolysaccharide-stimulated U937 human leukemia cells significantly increased the mRNA and protein levels of OCT4/NANOG preferentially in HBV-active (HBV+HBsAg+) HCC cells. The inflamed-CM also increased the side population (SP) cell percentage, green fluorescent protein (GFP)-positive cell population, and luciferase activity of OCT4 promoter-GFP/luciferase in HBV-active HCC cells. Furthermore, the inflamed-CM upregulated the expressions of insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) and activated IGF-IR/Akt signaling in HBV-HCC. The IGF-IR phosphorylation inhibitor picropodophyllin (PPP) suppressed inflamed-CM-induced OCT4 and NANOG levels in HBV+HBsAg+ Hep3B cells. Forced expression of OCT4 significantly increased the secondary sphere formation and cell migration, and reduced susceptibility of HBV-HCC cells to cisplatin, bleomycin, and doxorubicin. Taking together, our results show that niche inflammatory mediators play critical roles in inducing the expression of stemness-related properties involving IGF-IR activation, and

  7. All-trans retinoic acid decreases susceptibility of a gastric cancer cell line to lymphokine-activated killer cytotoxicity.

    PubMed Central

    Chao, T. Y.; Jiang, S. Y.; Shyu, R. Y.; Yeh, M. Y.; Chu, T. M.

    1997-01-01

    All-trans retinoic acid (RA) was previously shown to regulate the growth of gastric cancer cells derived from the cell line SC-M1. This study was designed to investigate the effect of RA on the sensitivity of SC-M1 cells to lymphokine-activated killer (LAK) activity. RA at the concentration range of 0.001-10 microM was shown to induce SC-M1 cells to exhibit resistance to LAK activity in a dose-dependent manner. A kinetics study indicated that a significantly increased resistance was detected after 2 days of co-culturing SC-M1 cells with RA and reached a maximum after 6 days of culture. Similar results were obtained from two other cancer cell lines: promyelocytic leukaemia HL-60 and hepatic cancer Hep 3B. A binding assay demonstrated that the binding efficacy between target SC-M1 cells and effector LAK cells was not altered by RA. Flow cytometric analyses revealed that RA exhibited no effect on the expression of cell surface molecules, including HLA class I and class II antigens, intercellular adhesion molecule-1 and -2, and lymphocyte function antigen-3. Cell cycle analysis revealed that culture of SC-M1 cells with RA resulted in an increase in G0/G1 phase and a decrease in S phase, accompanied by a decrease in cyclin A and cyclin B1 mRNA as determined by Northern blot analysis. Additionally, RA was shown to enhance the expression of retinoic acid receptor alpha (RAR alpha) in SC-M1 cells, and to have no effect on the expression of RARbeta or RARgamma. Taken together, these results indicate that RA can significantly increase gastric cancer cells SC-M1 to resist LAK cytotoxicity by means of a cytostatic effect through a mechanism relating to cell cycle regulation. The prevailing ideas, such as a decrease in effector to target cell binding, a reduced MHC class I antigen expression or an altered RARbeta expression, are not involved. Images Figure 4 Figure 5 PMID:9155047

  8. Bortezomib alleviates drug-induced liver injury by regulating CYP2E1 gene transcription

    PubMed Central

    PARK, WOO-JAE; KIM, SO-YEON; KIM, YE-RYUNG; PARK, JOO-WON

    2016-01-01

    Acute liver failure, i.e., the fatal deterioration of liver function, is the most common indication that emergency liver transplantation is necessary. Moreover, in the USA, drug-induced liver injury (DILI), including acetaminophen (APAP)-induced hepatotoxicity, is the main cause of acute liver failure. Matching a donor for liver transplantation is extremely difficult, and thus the development of a novel therapy for DILI is urgently needed. Following recent approval by the FDA of the proteasomal inhibitor bortezomib, its therapeutic effects on various human diseases, including solid and hematologic malignancies, have been validated. However, the specific action of proteasomal inhibition in cases of DILI had not been elucidated prior to this study. To examine the effects of proteasomal inhibition in DILI experimentally, male C56Bl/6 mice were injected with 1 mg bortezomib/kg before APAP treatment. Bortezomib not only alleviated APAP-induced hepatotoxicity in a time- and dose-dependent manner, it also alleviated CCl4- and thioacetamide-induced hepatotoxicity. We also noted that bortezomib significantly reduced cytochrome P450 2E1 (CYP2E1) expression and activity in the liver, which was accompanied by the induction of endoplasmic reticulum (ER) stress. In addition, bortezomib decreased hepatocyte nuclear factor-1α-induced promoter activation of CYP2E1 in Hep3B cells. By contrast, another proteasome inhibitor, MG132, did not cause ER stress and did not markedly affect CYP2E1 enzyme activity. Liver injury induced by APAP was aggravated by MG132, possibly via elevation of connexin 32 expression. This study suggests that proteasome inhibition has different effects in cases of DILI depending on the specific inhibitor being used. Furthermore, results from the mouse model indicated that bortezomib, but not MG132, was effective in alleviating DILI. ER stress induced by proteasome inhibition has previously been shown to exert various effects on DILI patients, and thus each

  9. Therapeutic role of a synthesized calcium phosphate nanocomposite material on hepatocarcinogenesis in rats.

    PubMed

    Mohammed, Magdy; Abdel-Gawad, Eman; Awwad, Sameh; Kandil, Eman; El-Agamy, Basma

    2016-06-01

    Nanotechnology research is booming worldwide, and the general belief is that medical and biological applications will form the greatest sector of expansion over the next decade. With this in mind, this study was designed to evaluate the therapeutic effects of a synthesized tricalcium phosphate nanocomposite material (nano-TCP) on hepatocarcinoma in a rat model, as initiated with diethylnitrosamine (DEN) and promoted with phenobarbital (PB). Hepatocarcinoma was induced with intraperitoneal injections of DEN (50 mg·(kg body mass)(-1)) 3 times a week for 2 weeks. Three weeks after the last dose of DEN, the rats received PB (0.05 %, w/v) in their drinking water for a further 6 weeks. Nano-TCP (100 mg·(kg body mass)(-1)) was administered intraperitoneally 3 times per week to rats with HCC. At the end of the experimental period, liver samples were collected from all animals for biochemical and histopathological analysis. The degree of DNA fragmentation was analyzed, in addition to immune status, by measuring the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2). The activities of the most important free-radical scavengers of the antioxidant defense system as well as malondialdehyde (MDA) content and liver enzymes were measured. The levels of hepatic heat shock protein-70 (HSP-70), caspase-3, and metalloproteinase-9 were also measured as markers for inflammation and apoptosis. Histopathological examination of liver tissue was performed. The results revealed the potent efficacy of nano-TCP in repairing the fragmented DNA and ameliorating most of the investigated parameters by significant elevation in the levels of hepatic alanine aminotransferase (ALT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. On the other hand, there was a significant decrease in hepatic gamma-glutamyl transpeptidase (γ-GT), MDA, IL-2, IFN-γ, TNF-α, matrix metalloproteinase-9 (MMP-9), HSP-70, and caspase-3 levels upon

  10. Synthesis, Evaluation of Anticancer Activity and QSAR Study of Heterocyclic Esters of Caffeic Acid

    PubMed Central

    Hajmohamad Ebrahim Ketabforoosh, Shima; Amini, Mohsen; Vosooghi, Mohsen; Shafiee, Abbas; Azizi, Ebrahim; Kobarfard, Farzad

    2013-01-01

    Caffeic acid phenethyl ester (CAPE) suppresses the growth of transformed cells such as human breast cancer cells, hepatocarcinoma , myeloid leukemia, colorectal cancer cells, fibrosarcoma, glioma and melanoma. A group of heterocyclic esters of caffeic acid was synthesized using Mitsunobu reaction and the esters were subjected to further structural modification by electrooxidation of the catechol ring of caffeic acid esters in the presence of sodium benzenesulfinate and sodium toluensulfinate as nucleophiles. Both heterocyclic esters of caffeic acid and their arylsulfonyl derivatives were evaluated for their cytotoxic activity against HeLa, SK-OV-3, and HT-29 cancer cell lines. HeLa cells showed the highest sensitivity to the compounds and heterocyclic esters with no substituent on catechol ring showed better activity compared to their substituted counterparts. QSAR studies reemphasized the importance of molecular shape of the compounds for their cytotoxic activity. PMID:24523750

  11. New trends in the multidisciplinary treatment of liver tumors.

    PubMed

    Donati, Marcello; Basile, Francesco

    2013-08-01

    The authors report on new trends in the multidisciplinary treatment of liver tumors from the 3rd Interdisciplinary Treatment of Liver Tumors Congress in Essen, Germany. Novel aspects of molecular target-oriented therapies of hepatocarcinoma were reported, such as the current position of radiofrequency and microwave ablation of tumors compared with the new electroporation technique. State-of-the-art and new surgical trends in cholangiocarcinoma treatment were also discussed. The correlation of the biological behavior involved in the differentiation of melanomas with the treatment of liver metastasis was better defined. The role of neoadjuvant chemotherapy and combined strategies for colorectal liver metastasis were focused on intra-arterial therapeutic options. New data on neuroendocrine tumor liver metastases were also shared, leading to a better orientation for patient selection for surgical treatment. PMID:23902241

  12. In vitro comparative analysis of antiproliferative activity of essential oil from mandarin peel and its principal component limonene.

    PubMed

    Manassero, Carlos A; Girotti, Juan R; Mijailovsky, Sergio; García de Bravo, Margarita; Polo, Mónica

    2013-01-01

    The effects of the essential oil of mandarin peel (Corrientes, Argentina) and limonene (its major component) were studied on two human tumour cell lines growth (lung adenocarcinoma A549 and hepatocarcinoma HepG2). The essential oil was obtained by cold press and its composition was investigated by gas chromatography (GC) and GC/mass spectrometry (MS) analysis. The antiproliferative effect was studied using an MTT assay. Both mandarin essential oil and limonene tested showed a strong dose-dependent effect on the growth inhibition of these cell lines. The essential oil was more effective in A549 than in HepG2 cells and more effective than limonene in both the cases. It is likely that minor components and limonene of the oil could exert additive or synergistic effects. Hence, mandarin essential oil could lead to the development of anti-tumour agent or complementary and alternative medicines for the treatment of diverse cancers. PMID:22943501

  13. Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

    PubMed

    Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

    2016-08-01

    Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. PMID:27113918

  14. A comparative evaluation of biological activities and bioactive compounds of the seagrasses Zostera marina and Zostera noltei from southern Portugal.

    PubMed

    Custódio, Luísa; Laukaityte, Simona; Engelen, Aschwin H; Rodrigues, Maria João; Pereira, Hugo; Vizetto-Duarte, Catarina; Barreira, Luísa; Rodríguez, Hortensia; Alberício, Fernando; Varela, João

    2016-01-01

    This work assessed the antioxidant potential, acetylcholinesterase (AChE) inhibition and the in vitro cytotoxic activity of extracts of the seagrasses Zostera marina and Zostera noltei collected from southern Portugal. The total phenolic contents (TPCs), the rosmarinic acid (RA) concentration (HPLC/DAD) and the fatty acid (FA) profile (GC/MS) are also described. Z. marina had the highest TPC, radical scavenging activity against DPPH radicals and copper chelating activity. Z. noltei had metal chelation capacity to copper and iron ions. None of the species was able to inhibit AChE. Both seagrasses had high levels of polyunsaturated FAs. Z. marina significantly and selectively reduced the viability of tumorous neuronal cells. Z. noltei was highly toxic for the three cell lines tested and was selective against hepatocarcinoma cells at the concentration of 100 μg/mL. RA was the main compound identified in Z. marina, but not in Z. noltei. PMID:26189828

  15. Synthesis of some p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-thiazoles and their anticancer activity.

    PubMed

    Zaharia, Valentin; Ignat, Adriana; Palibroda, Nicolae; Ngameni, Bathélémy; Kuete, Victor; Fokunang, Charles N; Moungang, Marlyse L; Ngadjui, Bonaventure T

    2010-11-01

    A series of novel p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-thiazoles derivatives (2a-f, 3a-f and 5-8) were synthesized by initial condensation of p-toluenesulfonylthiosemicarbazide 1 with a series of α-halogenocarbonyls in acetone or dimethylformamide (DMF)/acetone, mixture. All our synthesized compounds were submitted for further acylation reaction in the presence of acetic anhydride. The structures of newly synthesized derivatives 2a-f, 3a-f and 5-8 were confirmed by IR, (1)H-NMR, EIMS spectral data and elemental analysis. Compounds 2a, 2c, 2d, 2e and 3a showed significant anticancer activities (IC(50)<10 μM) on both prostate DU-145 and hepatocarcinoma Hep-G2 cancer cell lines. PMID:20810194

  16. In vitro and in vivo anticancer activity of surface modified paclitaxel attached hydroxyapatite and titanium dioxide nanoparticles.

    PubMed

    Venkatasubbu, G Devanand; Ramasamy, S; Reddy, G Pramod; Kumar, J

    2013-08-01

    Targeted drug delivery using nanocrystalline materials delivers the drug at the diseased site. This increases the efficacy of the drug in killing the cancer cells. Surface modifications were done to target the drug to a particular receptor on the cell surface. This paper reports synthesis of hydroxyapatite and titanium dioxide nanoparticles and modification of their surface with polyethylene glycol (PEG) followed by folic acid (FA). Paclitaxel, an anticancer drug, is attached to functionalized hydroxyapatite and titanium dioxide nanoparticles. The pure and functionalised nanoparticles are characterised with XRD, TEM and UV spectroscopy. Anticancer analysis was carried out in DEN induced hepatocarcinoma animals. Biochemical, hematological and histopathological analysis show that the surface modified paclitaxel attached nanoparticles have an higher anticancer activity than the pure paclitaxel and surface modified nanoparticles without paclitaxel. This is due to the targeting of the drug to the folate receptor in the cancer cells. PMID:23615724

  17. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells

    PubMed Central

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K.; Das, Mahua R.; Sen, Shamik; Sarkar, Debi P.; Jana, Siddhartha S.

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (−) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  18. [The use of ultrasonically-guided needle aspiration cytology in the diagnosis of hepatic lesions].

    PubMed

    Geroni, P L; Caffi Avogadri, G M; Costardi, F

    1990-10-31

    The paper reports the results of 68 ultrasonically-guided fine needle aspirations of the liver. Histological confirmation was carried out in 24 cases and in 2 of these there was discrepancy with regard to the type of malignant neoplasia (hepatocarcinoma versus metastasis following adenocarcinoma). Following a discussion of the advantages and disadvantages of ultrasonically-guided fine needle aspiration of hepatic lesions in comparison to other techniques, including CT, RNM, angiography, laparoscopy and scintigraphy, the specificity, sensitivity and diagnostic accuracy of the method are determined. Particular emphasis is placed on the ease of performance, the possibility of reaching concealed targets, the limited nature of complications and the reduced cost. The Authors conclude that this technique should be preferentially used in the diagnosis of nodular hepatic lesions. PMID:2082208

  19. Antiproliferative activity and apoptosis inducing effects of nitric oxide donating derivatives of evodiamine.

    PubMed

    Zhao, Nan; Tian, Kang-Tao; Cheng, Ke-Guang; Han, Tong; Hu, Xu; Li, Da-Hong; Li, Zhan-Lin; Hua, Hui-Ming

    2016-07-01

    The first series of nitric oxide donating derivatives of evodiamine were designed and prepared. NO releasing ability of all target derivatives was evaluated in BGC-823, Bel-7402 and L-02 cells. The cytotoxicity was evaluated against three human tumor cell lines (Bel-7402, A549 and BGC-823) and normal human liver cells L-02. The nitrate derivatives 11a and 11b only exhibited moderate activity and furoxan-based derivatives 13a-c, 14a and 14b showed promising activity. 13c showed good cytotoxic selectivity between tumor and normal liver cells and was further investigated for its apoptotic properties on human hepatocarcinoma Bel-7402 cells. The molecular mode of action revealed that 13c caused cell-cycle arrest at S phase and induced apoptosis in Bel-7402 cells through mitochondria-related caspase-dependent pathways. PMID:27178387

  20. Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Chen, Haimin; Yan, Xiaojun; Zheng, Yanling

    2013-05-01

    This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosisinducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.

  1. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

    PubMed

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K; Das, Mahua R; Sen, Shamik; Sarkar, Debi P; Jana, Siddhartha S

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  2. Estrogen Represses Hepatocellular Carcinoma (HCC) Growth via Inhibiting Alternative Activation of Tumor-associated Macrophages (TAMs)*

    PubMed Central

    Yang, Weiwei; Lu, Yan; Xu, Yichen; Xu, Lizhi; Zheng, Wei; Wu, Yuanyuan; Li, Long; Shen, Pingping

    2012-01-01

    Hepatocarcinoma cancer (HCC), one of the most malignant cancers, occurs significantly more often in men than in women; however, little is known about its underlying molecular mechanisms. Here we identified that 17β-estradiol (E2) could suppress tumor growth via regulating the polarization of macrophages. We showed that E2 re-administration reduced tumor growth in orthotopic and ectopic mice HCC models. E2 functioned as a suppressor for macrophage alternative activation and tumor progression by keeping estrogen receptor β (ERβ) away from interacting with ATP5J (also known as ATPase-coupling factor 6), a part of ATPase, thus inhibiting the JAK1-STAT6 signaling pathway. These studies introduce a novel mechanism for suppressing male-predominant HCC. PMID:22908233

  3. Selective inhibition of liver cancer growth realized by the intrinsic toxicity of a quantum dot–lipid complex

    PubMed Central

    Shao, Dan; Li, Jing; Guan, Fengying; Pan, Yue; Xiao, Xuanang; Zhang, Ming; Zhang, Hong; Chen, Li

    2014-01-01

    Using the intrinsic toxicity of nanomaterials for anticancer therapy is an emerging concept. In this work, we discovered that CdTe/CdS quantum dots, when coated with lipids (QD-LC) instead of popular liposomes, polymers, or dendrimers, demonstrated extraordinarily high specificity for cancer cells, which was due to the difference in the macropinocytosis uptake pathways of QD-LC between the cancer cells and the normal cells. QD-LC-induced HepG2 cell apoptosis was concomitant with the activation of the JNK/caspase-3 signaling pathway. Moreover, QD-LC treatment resulted in a delay in the latent period for microtumor formation of mouse hepatocarcinoma H22 cells and inhibited tumor growth, with a reduction of 53.2% in tumor volume without toxicity in major organs after intratumoral administrations to tumor-bearing mice. Our results demonstrate that QD-LC could be a very promising theranostic agent against liver cancer. PMID:25525357

  4. Fumigaclavine I, a new alkaloid isolated from endophyte Aspergillus terreus.

    PubMed

    Shen, Li; Zhu, Li; Luo, Qian; Li, Xiao-Wen; Xi, Ju-Qun; Kong, Gui-Mei; Song, Yong-Chun

    2015-12-01

    The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I (1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C (2), rhizoctonic acid (3), monomethylsulochrin (4), chaetominine (5), spirotryprostatin A (6), asperfumoid (7), and lumichrome (8). The structure of compound 1 was elucidated by various spectroscopic analyses (UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent. PMID:26721713

  5. Spherically-clustered porous Au-Ag alloy nanoparticle prepared by partial inhibition of galvanic replacement and its application for efficient multimodal therapy.

    PubMed

    Jang, Hongje; Min, Dal-Hee

    2015-03-24

    The polyvinylpyrrolidone (PVP)-coated spherically clustered porous gold-silver alloy nanoparticle (PVP-SPAN) was prepared by low temperature mediated, partially inhibited galvanic replacement reaction followed by silver etching process. The prepared porous nanostructures exhibited excellent photothermal conversion efficiency under irradiation of near-infrared light (NIR) and allowed a high payload of both doxorubicin (Dox) and thiolated dye-labeled oligonucleotide, DNAzyme (FDz). Especially, PVP-SPAN provided 10 times higher loading capacity for oligonucleotide than conventional hollow nanoshells due to increased pore diameter and surface-to-volume ratio. We demonstrated highly efficient chemo-thermo-gene multitherapy based on codelivery of Dox and FDz with NIR-mediated photothermal therapeutic effect using a model system of hepatitis C virus infected human liver cells (Huh7 human hepatocarcinoma cell line containing hepatitis C virus NS3 gene replicon) compared to conventional hollow nanoshells. PMID:25560916

  6. In Situ Biosynthesis of Fluorescent Platinum Nanoclusters: Toward Self-Bioimaging-Guided Cancer Theranostics.

    PubMed

    Chen, Donghua; Zhao, Chunqiu; Ye, Jing; Li, Qiwei; Liu, Xiaoli; Su, Meina; Jiang, Hui; Amatore, Christian; Selke, Matthias; Wang, Xuemei

    2015-08-19

    Among the noble-metal clusters, very few reports about platinum clusters were used as bioimaging probes of tumors except as a reducing catalyst. It is first established herein that the biocompatible platinum nanoclusters are spontaneously biosynthesized by cancerous cells (i.e., HepG2 (human hepatocarcinoma), A549 (lung cancer), and others) rather than noncancerous cells (i.e., L02 (human embryo liver cells)) when incubated with micromolar chloroplatinic acid solutions. These in situ biosynthesized platinum nanoclusters could be readily realized in a biological environment and emit a bright fluorescence at 460 nm, which could be further utilized to facilitate an excellent cancer-cell-killing efficiency when combined with porphyrin derivatives for photothermal treatment. This raises the possibility of providing a promising and precise bioimaging strategy for specific fluorescent self-biomarking of tumor locations and realizing fluorescence imaging-guided photothermal therapy of tumors. PMID:26227621

  7. Enhanced removal of COD and color from landfill leachate in a sequential bioreactor.

    PubMed

    Ghosh, Pooja; Swati; Thakur, Indu Shekhar

    2014-10-01

    In the present study, a sequential treatment process was carried out using a fungal sp. (Phanerochaete sp.) followed by a bacterial sp. (Pseudomonas sp.) for the degradation and detoxification of contaminants in landfill leachate. The process was optimized using Box-Behnken design (BBD) and response surface methodology (RSM) for three variables (C source, N source and duration), while monitoring two responses (% COD and color removal). After treatment in a bioreactor under optimized conditions, enhanced removal of COD (76.9%) and color (45.4%) was observed. Further, GC-MS analysis of metabolites detected at different stages of treatment showed formation of degradation products of lignin and polycyclic aromatic compounds. Treatment efficiency was finally evaluated by the alkaline comet assay in HepG2 human hepato-carcinoma cells. The results indicated no statistically significant DNA damage at the end of the treatment, making the effluent suitable to be discharged conforming to the safety standards. PMID:25113402

  8. Potential Use of Biological Proteins for Liver Failure Therapy

    PubMed Central

    Taguchi, Kazuaki; Yamasaki, Keishi; Seo, Hakaru; Otagiri, Masaki

    2015-01-01

    Biological proteins have unlimited potential for use as pharmaceutical products due to their various biological activities, which include non-toxicity, biocompatibility, and biodegradability. Recent scientific advances allow for the development of novel innovative protein-based products that draw on the quality of their innate biological activities. Some of them hold promising potential for novel therapeutic agents/devices for addressing hepatic diseases such as hepatitis, fibrosis, and hepatocarcinomas. This review attempts to provide an overview of the development of protein-based products that take advantage of their biological activity for medication, and discusses possibilities for the therapeutic potential of protein-based products produced through different approaches to specifically target the liver (or hepatic cells: hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) in the treatment of hepatic diseases. PMID:26404356

  9. Enantioselective apoptosis induced by individual isomers of bifenthrin in Hep G2 cells.

    PubMed

    Liu, Huigang; Li, Juan

    2015-03-01

    Bifenthrin (BF) has been used in racemate for agricultural purposes against soil insects, leading to increased inputs into soil environments. However, most of the studies about the toxicology research on BF were performed in its racemic form. The aim of the present study was to evaluate the enantiomer-specific cis-BF-induced apoptosis and intracellular reactive oxygen species (ROS) generation on human hepatocarcinoma cells (Hep G2). The results of cell viability assay and cytoflow assay indicated an obvious enantioselective hepatocyte toxicity of 1S-cis-BF in Hep G2 cells. 1S-cis-BF also induced ROS production, up-regulated Bax protein expression and down-regulated Bcl-2 expression levels. The present study suggested that enantioselective toxicity should be evaluated on currently used chiral pesticides, such as synthetic pyrethroids. PMID:25748094

  10. Long-Term Placement of Subcutaneous Ruesch-Type Stents for Double Biliary Stenosis in a Living-Donor Liver Transplant Recipient

    SciTech Connect

    Adani, Gian Luigi Baccarani, Umberto; Lorenzin, Dario; Risaliti, Andrea; Como, Giuseppe; Gasparini, Daniele; Sponza, Massimo; Bresadola, Vittorio; Bresadola, Fabrizio

    2007-04-15

    Biliary reconstruction continues to be a major source of morbidity following liver transplantation. The spectrum of biliary complications is evolving due to the increasing number of split-liver and living-donor liver transplantation, which are even associated with a higher incidence of biliary complications. Bile duct strictures are the most common cause of late biliary complications and account for up to 40% of all biliary complications. Optimal therapy for posttransplantation anastomotic biliary strictures remains uncertain and requires a multidisciplinary approach. We report the case of a 54-year-old Caucasian male affected by hepatocarcinoma and hepatitis C-related cirrhosis who underwent right-lobe living-donor liver transplantation from his son complicated by double anastomotic stenosis of the main right hepatic duct and of an accessory biliary duct draining segments 6 and 7 of the graft that was successfully treated by percutaneous transhepatic cholangiography with long-term subcutaneous placement of two internal Ruesch-type biliary stents.

  11. Metabolic Activation of Rhein: Insights into the Potential Toxicity Induced by Rhein-Containing Herbs.

    PubMed

    Yuan, Yuan; Zheng, Jiyue; Wang, Meiyu; Li, Yuan; Ruan, Jianqing; Zhang, Hongjian

    2016-07-20

    Rhein is a major component of the many medicinal herbs such as rhubarb. Despite wide use, intoxication cases associated with rhein-containing herbs are often reported. The present work aimed to investigate if rhein was subject to metabolic activation leading to toxicity. Upon incubations with different species of liver microsomes, three monoglucuronides were identified, corresponding to two hydroxyl glucuronides and one acyl glucuronide via the carboxyl group, respectively. Further study revealed that rhein acyl glucuronide was chemically reactive, and showed cytotoxicity toward hepatocarcinoma cells. In addition, significant species differences in glucuronidation of rhein were observed between laboratory animals and humans. Reaction phenotyping experiments demonstrated that rhein acyl glucuronide was catalyzed predominantly by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A9, and 2B7. Taken together, the present study confirmed that rhein could be metabolically activated via the formation of acyl glucuronide, especially in human. PMID:27362917

  12. The aryl hydrocarbon receptor agonist benzo(a)pyrene reactivates LINE-1 in HepG2 cells through canonical TGF-β1 signaling: implications in hepatocellular carcinogenesis

    PubMed Central

    Reyes-Reyes, Elsa M; Ramos, Irma N; Tavera-Garcia, Marco A; Ramos, Kenneth S

    2016-01-01

    Long interspersed nuclear element-1 (L1) is a genetic element that mobilizes throughout the mammalian genome via retrotransposition and damages host DNA via mutational insertions, chromosomal rearrangements, and reprogramming of gene expression. The cellular mechanisms responsible for aberrant L1 expression during cancer pathogenesis are unclear. Previously, we have shown that L1 reactivation in several human cell lines is dependent upon the activation of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor member of the PAS superfamily of proteins. We also showed that ectopic expression of L1 reprograms the HepG2 genome leading to epithelial-to-mesenchymal transition (EMT). Here we present evidence that reactivation of L1 and modulation of EMT in HepG2 cells by the AhR ligand benzo(a)pyrene (BaP) is effected through the canonical TGF-β1 signaling pathway. BaP increased TGF-β1 mRNA, SMAD2 phosphorylation and decreased expression of E-Cadherin. The functional relevance of these interactions and the involvement of TGFBR1/ALK5 and SMAD2/3 were confirmed by siRNA interference. Furthermore, expression of L1-encoded ORF1p was positively correlated with the activation of TGF-β1 signaling in human hepatocarcinoma samples at various stages of malignant progression. These results indicate that ligand-mediated AhR activation regulates L1 via canonical TGF-β1 signaling and raise important questions about the molecular etiology of human hepatocarcinomas. PMID:27293999

  13. Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

    SciTech Connect

    Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu

    2014-02-28

    Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.

  14. DESAFÍOS ÉTICOS DE LA INVESTIGACIÓN CON ANIMALES, MANIPULACIÓN GENÉTICA

    PubMed Central

    Yunta, Eduardo Rodríguez

    2012-01-01

    En la investigación con animales existen cuestionamientos éticos tanto en el uso como modelos de enfermedades humanas y requisito previo para ensayos en humanos como en la introducción de modificaciones genéticas. Algunos de estos cuestionamientos son: no representar exactamente la condición humana como modelos, realizar pruebas de toxicidad con grave daño para los animales, alterar su naturaleza mediante modificaciones genéticas, riesgos de la introducción de organismos genéticamente modificados. El uso de animales en investigación para beneficio humano, impone al ser humano la responsabilidad moral de respetarlo, no haciéndoles sufrir innecesariamente, al estar trabajando con seres vivientes y sentientes. PMID:23338641

  15. Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

    PubMed Central

    Thibault, Patricia A.; Huys, Adam; Amador-Cañizares, Yalena; Gailius, Julie E.; Pinel, Dayna E.

    2015-01-01

    ABSTRACT miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5′ untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound full-length RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5′-to-3′ exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122. IMPORTANCE The functions of miR-122 in the promotion of the HCV life cycle are not fully understood. Here, we show that binding of miR-122 to each of the two binding sites in the HCV 5′ UTR contributes equally to HCV replication and that binding to both sites can function cooperatively. This suggests that active Ago2–miR-122 complexes

  16. Ciencia en la Estación Espacial Internacional

    NASA Video Gallery

    Durante más de 10 años, los seres humanos vivieron en la estación espacial las 24 horas, los siete días de la semana Y llevaron a cabo más de 600 experimentos. Fíjate apenas algunos de estos experi...

  17. Dos dosis de vacuna contra los VPH pueden proteger

    Cancer.gov

    Dos dosis de Cervarix, la vacuna contra virus del papiloma humano (VPH), fueron tan efectivas como la pauta normal actual de tres dosis después de cuatro años de seguimiento. El estudio de vacuna en Costa Rica, patrocinado por el NCI, fue diseñado para ev

  18. Harold Varmus investido bajo juramento como 14.º director d

    Cancer.gov

    Ganador del Premio Nobel, doctor Harold E. Varmus, prestó juramento hoy como 14.º director del Instituto Nacional del Cáncer (NCI).  "Es muy estimulante que estés de regreso con nosotros", dijo la secretaria del Departamento de Salud y Servicios Humanos K

  19. Disminuyen en los Estados Unidos las infecciones por VPH.

    Cancer.gov

    La infección por los tipos del virus del papiloma humano (VPH) en el blanco de la vacuna cuadrivalente se redujo en casi dos tercios en las adolescentes desde que se recomendó la vacunación en los Estados Unidos.

  20. Robonauta 2

    NASA Video Gallery

    Casi 200 personas de 15 países han visitado la Estación Espacial Internacional, pero, hasta ahora, la órbita sólo tuvo humanos como miembros de la tripulación. Robonauta 2, la última generación de ...

  1. [Bio-nanoscience and bio-nanotechnology: social and ethical aspects].

    PubMed

    Martín-Lomas, Manuel

    2006-01-01

    This article is an abstract of a conference with the same title presented at the XIII Jornadas sobre Derecho y Genoma Humano and is basically centred in a report for the Royal Society and the Royal Academy of Engineering entitled Nanoscience and Nanotechnology made publicly available July 2004. PMID:17393798

  2. New trends in guided nanotherapies for digestive cancers: A systematic review.

    PubMed

    Fernandes, Elisabete; Ferreira, José Alexandre; Andreia, Peixoto; Luís, Lima; Barroso, Sérgio; Sarmento, Bruno; Santos, Lúcio Lara

    2015-07-10

    Digestive tract tumors are among the most common and deadliest malignancies worldwide, mainly due to late diagnosis and lack of efficient therapeutics. Current treatments essentially rely on surgery associated with (neo)adjuvant chemotherapy agents. Despite an upfront response, conventional drugs often fail to eliminate highly aggressive clones endowed with chemoresistant properties, which are responsible for tumor recurrence and disease dissemination. Synthetic drugs also present severe adverse systemic effects, hampering the administration of biologically effective dosages. Nanoencapsulation of chemotherapeutic agents within biocompatible polymeric or lipid matrices holds great potential to improve the pharmacokinetics and efficacy of conventional chemotherapy while reducing systemic toxicity. Tagging nanoparticle surfaces with specific ligands for cancer cells, namely monoclonal antibodies or antibody fragments, has provided means to target more aggressive clones, further improving the selectivity and efficacy of nanodelivery vehicles. In fact, over the past twenty years, significant research has translated into a wide array of guided nanoparticles, providing the molecular background for a new generation of intelligent and more effective anti-cancer agents. Attempting to bring awareness among the medical community to emerging targeted nanopharmaceuticals and foster advances in the field, we have conducted a systematic review about this matter. Emphasis was set on ongoing preclinical and clinical trials for liver, colorectal, gastric and pancreatic cancers. To the best of our knowledge this is the first systematic and integrated overview on this field. Using a specific query, 433 abstracts were gathered and narrowed to 47 manuscripts when matched against inclusion/exclusion criteria. All studies showed that active targeting improves the effectiveness of the nanodrugs alone, while lowering its side effects. The main focus has been on hepatocarcinomas, mainly by

  3. NO-Releasing Enmein-Type Diterpenoid Derivatives with Selective Antiproliferative Activity and Effects on Apoptosis-Related Proteins.

    PubMed

    Li, Dahong; Hu, Xu; Han, Tong; Liao, Jie; Xiao, Wei; Xu, Shengtao; Li, Zhanlin; Wang, Zhenzhong; Hua, Huiming; Xu, Jinyi

    2016-01-01

    A series of nine enmein-type ent-kaurane diterpenoid and furoxan-based nitric oxide (NO) donor hybrids (10a-i) were designed and synthesized from commercially available oridonin (1). These hybrids were evaluated for their antiproliferative activity against Bel-7402, K562, MGC-803, and CaEs-17 human cancer cell lines and L-02 normal liver cells. The antiproliferative activity against tumor cells was stronger than the lead compound 1 and parent molecule 9 in most cases. Especially, compound 10f showed the strongest activity against human hepatocarcinoma Bel-7402 cell line with an IC50 of 0.81 μM and could also release 33.7 μmol/L NO at the time point of 60 min. Compounds 10a-i also showed cytotoxic selectivity between tumor and normal liver cells with IC50 ranging from 22.1 to 33.9 μM. Furthermore, the apoptotic properties on Bel-7402 cells revealed that 10f could induce S phase cell cycle arrest and apoptosis at low micromolar concentrations. The effects of 10f on apoptosis-related proteins were also investigated. The potent antiproliferative activities and mechanistic studies warrant further preclinical investigations. PMID:27617998

  4. Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

    PubMed Central

    Diaz, Rosario; Luengo-Arratta, Sandra A.; Seixas, João D.; Amata, Emanuele; Devine, William; Cordon-Obras, Carlos; Rojas-Barros, Domingo I.; Jimenez, Elena; Ortega, Fatima; Crouch, Sabrinia; Colmenarejo, Gonzalo; Fiandor, Jose Maria; Martin, Jose Julio; Berlanga, Manuela; Gonzalez, Silvia; Manzano, Pilar; Navarro, Miguel; Pollastri, Michael P.

    2014-01-01

    In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs. PMID:25340575

  5. Chelerythrine and Fe3O4 Loaded Multi-Walled Carbon Nanotubes for Targeted Cancer Therapy.

    PubMed

    Cao, Liangli; Liang, Yongbo; Zhao, Feijun; Zhao, Xiongjie; Chen, Zhencheng

    2016-06-01

    The work focused on manufacturing improved drug loaded multifunctional magnetic nanoparticles that can overcome the relative non-specificity and potential side-effects of some chemotherapeutic drugs to healthy tissues. A new drug delivery system, Chelerythrine (CHE) and Fe3O4 loaded multi-walled carbon nanotubes (Fe3O4/MWNTs-CHE nanocomposites) that can target hepatocytes when treating malignant tumors, was prepared through a simple adsorption method. The formulation and structure of the Fe3O4/MWNTs-CHE nanocomposites were characterized by vibrating sample magnetometer (VSM), Fourier Transform infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The cytotoxicity and anti-proliferation effect from the prepared nanocomposites were in vitro tested on human hepatocarcinoma HepG2 and normal liver LO2 cell lines. The results showed the saturated magnetization of Fe3O4/MWNTs-CHE nanocomposites could reach to 45.4O3 emu/g, and the in vitro CHE release behavior exhibited a biphasic release pattern. Moreover, the in vitro cytotoxicity studies revealed that the Fe3O4/MWNTs-CHE nanocomposites showed an efficient inhibition rate to HepG2 cell line and exhibited a lower cytotoxicity to LO2 cell line in comparison to the native CHE. Therefore, the multifunctional Fe3O4/MWNTs-CHE nanocomposites should be a useful and promising candidate for treatment of malignant tumors. PMID:27319224

  6. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    PubMed Central

    Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  7. [Current status of estrogen-progestational contraception].

    PubMed

    Tabaste, J L; Lesourd-Pontonnier, F

    1994-01-01

    The final decade of the century confirms the importance of latest generation progestogens in terms of decreased risks associated with the use of doses of ethinyl estradiol of 50 gamma or more, equally effective in contraception of this type. These new combination very considerably limit the untoward effects seen in the past regarding carbohydrate and lipid metabolism, as well as cardiovascular disease. Concerning carcinogenesis, the long term protective effect against carcinomas of the endometrium and ovary is now accepted, while studies of breast cancer are not unanimous in their condemnation, the same applying to the risk of hepatocarcinoma. Regulation of cycles is satisfactory and the vaginal flora little affected. Moderate and stable fibroids seem to be well controlled, the same applying to mucosal hyperplasia. Pregnancy, particularly rare, is not influenced unfavourably and the effect on lactation appears to be nil. Provided that regular monitoring is ensured and contraindications are respected, age does not appear to be an absolute limiting factor to its use but the risk of concomitant smoking remains. PMID:7973341

  8. HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2

    PubMed Central

    Granato, M.; Zompetta, C.; Vescarelli, E.; Rizzello, C.; Cardi, A.; Valia, S.; Antonelli, G.; Marchese, C.; Torrisi, M. R.; Faggioni, A.; Cirone, M.

    2016-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis. PMID:27476557

  9. pH-Responsive Hyaluronic Acid-Based Mixed Micelles for the Hepatoma-Targeting Delivery of Doxorubicin

    PubMed Central

    Wu, Jing-Liang; Tian, Gui-Xiang; Yu, Wen-Jing; Jia, Guang-Tao; Sun, Tong-Yi; Gao, Zhi-Qin

    2016-01-01

    The tumor targetability and stimulus responsivity of drug delivery systems are crucial in cancer diagnosis and treatment. In this study, hepatoma-targeting mixed micelles composed of a hyaluronic acid–glycyrrhetinic acid conjugate and a hyaluronic acid-l-histidine conjugate (HA–GA/HA–His) were prepared through ultrasonic dispersion. The formation and characterization of the mixed micelles were confirmed via 1H-NMR, particle size, and ζ potential measurements. The in vitro cellular uptake of the micelles was evaluated using human liver carcinoma (HepG2) cells. The antitumor effect of doxorubicin (DOX)-loaded micelles was investigated in vitro and in vivo. Results indicated that the DOX-loaded HA–GA/HA–His micelles showed a pH-dependent controlled release and were remarkably absorbed by HepG2 cells. Compared with free DOX, the DOX-loaded HA–GA/HA–His micelles showed a higher cytotoxicity to HepG2 cells. Moreover, the micelles effectively inhibited tumor growth in H22 cell-bearing mice. These results suggest that the HA–GA/HA–His mixed micelles are a good candidate for drug delivery in the prevention and treatment of hepatocarcinoma. PMID:27043540

  10. Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo.

    PubMed

    Jiang, Zhiwen; Han, Baoqin; Li, Hui; Yang, Yan; Liu, Wanshun

    2015-09-20

    Carboxymethyl chitosan (CMCS), with potent water solubility, biocompatibility, and non-toxicity, has emerged as a promising candidate for biomedical applications. In this study, the anti-tumor angiogenesis effects of CMCS were evaluated in vitro and in vivo. Our results showed that CMCS could inhibit the 2-dimensional and 3-dimensional migration of human umbilical vein endothelial cells (HUVECs) in vitro. CMCS significantly inhibited the growth of mouse hepatocarcinoma 22 tissues and could promote tumor cell necrosis as suggested by pathological observations. The CD34 expression in H22 tumor tissue, the levels of vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 in serum was regulated by CMCS treatment. CMCS could significantly improve thymus index, spleen index, tumor necrosis factor α and interferon γ level. In a conclusion, CMCS possessed potent anti-tumor effects by inhibiting tumor angiogenesis, stimulating immune functions. Our date provide more foundation for application of CMCS in biomedicine or biomaterials for targeted anticancer drugs delivery. PMID:26050881

  11. [Acute leukemia in childhood: present status of 100 cases after 7 years of complete remission (author's transl)].

    PubMed

    Schaison, G; Jacquillat, C; Lemercier, N; Weil, M; Alby, N; Auclerc, M F; Boiron, M; Bernard, J

    1980-01-01

    Amongst 1,200 leukemie children treated between 1958 and 1971, 60 are in complete remission for more than 10 years and 100 for more than 7 years. There were 96 acute lymphoid and 4 acute myeloid leukemias. Ten patients who have relapsed in the past have not done so lately. The F/M sex ratio is 1.5. Poor prognostic features were initially absent in 2/3 of cases. In 1/3 there was associated hyperleucocytosis and/or tumours. 93 children are in remission, their treatment having been stopped for 1 to 12 years. Five children relapsed and 4 are in a second remission for more than 2 years. Two children died in remission: one from a hepatocarcinoma and one from cardiac failure. These patients have been shown to have the following: 1) normal growth; 2) normal puberty: 8 patients have been able to reproduce, giving 10 children, one with multiple malformations; 3) school achievement and later socioprofessional behaviour has been normal. The patients have often sought a medical or paramedical career. Sequelae are minimal, psychological problems being minimal in the child. With the protocols used, mean remission curve shows a plateau after 9 years and complete definitive care is achieved in 92 per cent of patients surviving at 7 years. The very distant future outlook is not known. No other malignant haematological disease has occurred but one child died from a carcinoma. PMID:6931627

  12. Thio-dimethylarsinate is a common metabolite in urine samples from arsenic-exposed women in Bangladesh

    SciTech Connect

    Raml, Reingard; Rumpler, Alice; Goessler, Walter; Vahter, Marie; Li Li; Ochi, Takafumi; Francesconi, Kevin A.

    2007-08-01

    Over the last 6 years, much work on arsenic species in urine samples has been directed toward the determination of the reduced dimethylated arsenic species, DMA(III), because of its high toxicity and perceived key role in the metabolism of inorganic arsenic. Recent work, however, has suggested that DMA(III) may at times have been misidentified because its chromatographic properties can be similar to those of thio-dimethylarsinate (thio-DMA). We analyzed by HPLC-ICPMS (inductively coupled plasma mass spectrometry) urine samples from 75 arsenic-exposed women from Bangladesh with total arsenic concentrations ranging from 8 to 1034 {mu}g As/L and found that thio-DMA was present in 44% of the samples at concentrations ranging mostly from trace amounts to 24 {mu}g As/L (one sample contained 123 {mu}g As/L). Cytotoxicity testing with HepG2 cells derived from human hepatocarcinoma indicated that thio-DMA was about 10-fold more cytotoxic than dimethylarsinate (DMA). The widespread occurrence of thio-DMA in urine from these arsenic-exposed women suggests that this arsenical may also be present in other urine samples and has so far escaped detection. The work highlights the need for analytical methods providing specific determinations of arsenic compounds in future studies on arsenic metabolism and toxicology.

  13. Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

    PubMed Central

    Dorsey, W. C.; Ford, B. D.; Roane, L.; Haynie, D. T.; Tchounwou, P. B.

    2005-01-01

    Ultra–wideband (UWB) technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR) could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM) cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5–20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8–24 h post exposure. UWBR exerted a statistically significant (p < 0.05) dose-dependent response in cell viability in both serum-treated and serum free medium (SFM) -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma. PMID:16705798

  14. Intracellular GSH Alterations and Its Relationship to Level of Resistance following Exposure to Cisplatin in Cancer Cells

    PubMed Central

    Jamali, Bardia; Nakhjavani, Maryam; Hosseinzadeh, Leila; Amidi, Salimeh; Nikounezhad, Nastaran; H. Shirazi, Farshad

    2015-01-01

    One of the major complications in cancer chemotherapy with cisplatin as one of the important medicines in treatment regimens of different cancers is the development of resistance. One of the most described cellular defense mechanisms involved in resistance is glutathione (GSH), thus in this study, the effects of cisplatin on the total intracellular GSH level (GSHi) in some sensitive and resistant variants of human cell lines (hepatocarcinoma HepG2, skin A375, cisplatin sensitive glioblastoma U373MG and cisplatin resistant glioblastoma U373MGCP, cisplatin sensitive ovary A2780S and cisplatin resistant A2780CP cells) were studied. MTT assay was performed to measure cytotoxicity of cisplatin (33.3 µM for 1 hour). Following cisplatin exposure, GSHi (per million cells) was evaluated using a photometrical assay up to 90 minutes. Our results indicate that there are significant differences between GSHi content of A2780CP and U373MGCP cells compared to other cell lines. Moreover, IC50 of cisplatin in different cells seems to have a relation with mean of GSH level in 90 minutes (GSH (mean)90). As a conclusion, it seems that resistance to cisplatin in different cell lines is more related with the diverse patterns of GSHi variations following cisplatin exposure than its original level, and/or its cellular increase or decrease. It is also suggested that GSH (mean)90 may be used as a factor for the prediction of cellular resistance to cisplatin. PMID:25901159

  15. Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways.

    PubMed

    Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

    2016-12-01

    Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis. PMID:27075340

  16. Is there a role for antioxidants in the prevention of infection-associated carcinogenesis and in the treatment of infection-driven tumors?

    PubMed

    De Luca, Chiara; Kharaeva, Zaira; Korkina, Liudmila

    2015-01-01

    Causative connections between infections and cancer are ascertained for several types of viruses, bacteria, and parasites. The mechanisms of cancer induction in chronically infected inflamed tissues strongly implicate oxygen- and nitrogen-centered reactive species, and an impairment of redox-sensitive molecular pathways involved in the tumorigenic transformation, tumor growth, altered immune defense, and in the mechanisms of tumor cell death and survival. Here, we briefly reviewed mechanistic data on carcinogenesis and tumor progression of three major infection-associated tumors, human papillomavirus-induced cervical cancer, hepatitis B virus-positive hepatocarcinoma, and Helicobacter pylori-positive gastric cancer. Notwithstanding the contradictory results of clinical studies on cancer chemoprevention with long-term, high dosage antioxidant vitamin/micronutrient supplementation, natural and synthetic agents with proven capacity to affect redox-dependent molecular pathways still hold the promise for preventing/delaying carcinogenesis initiation, as well as the overt malignancy evolution from dysplastic/ aplastic stages. Novel directions for a targeted antioxidant-based approach to the reduction of persistent infection-driven cancer risk stems from the current knowledge of critical factors in the host-microbe interaction leading to oncogenesis. An emerging role of redox active substances in the chemotherapy of tumors relies on their stimulating effects towards TRAIL-related apoptosis and the induction of intracellular oxidative stress. PMID:25496269

  17. Kuppfer Cells Trigger Nonalcoholic Steatohepatitis Development in Diet-induced Mouse Model through Tumor Necrosis Factor-α Production*

    PubMed Central

    Tosello-Trampont, Annie-Carole; Landes, Susan G.; Nguyen, Virginia; Novobrantseva, Tatiana I.; Hahn, Young S.

    2012-01-01

    Nonalcoholic steatohepatitis (NASH), characterized by lipid deposits within hepatocytes (steatosis), is associated with hepatic injury and inflammation and leads to the development of fibrosis, cirrhosis, and hepatocarcinoma. However, the pathogenic mechanism of NASH is not well understood. To determine the role of distinct innate myeloid subsets in the development of NASH, we examined the contribution of liver resident macrophages (i.e. Kupffer cells) and blood-derived monocytes in triggering liver inflammation and hepatic damage. Employing a murine model of NASH, we discovered a previously unappreciated role for TNFα and Kupffer cells in the initiation and progression of NASH. Sequential depletion of Kupffer cells reduced the incidence of liver injury, steatosis, and proinflammatory monocyte infiltration. Furthermore, our data show a differential contribution of Kupffer cells and blood monocytes during the development of NASH; Kupffer cells increased their production of TNFα, followed by infiltration of CD11bintLy6Chi monocytes, 2 and 10 days, respectively, after starting the methionine/choline-deficient (MCD) diet. Importantly, targeted knockdown of TNFα expression in myeloid cells decreased the incidence of NASH development by decreasing steatosis, liver damage, monocyte infiltration, and the production of inflammatory chemokines. Our findings suggest that the increase of TNFα-producing Kupffer cells in the liver is crucial for the early phase of NASH development by promoting blood monocyte infiltration through the production of IP-10 and MCP-1. PMID:23066023

  18. Radio-frequency ablation-based studies on VX2rabbit models for HCC treatment.

    PubMed

    Bimonte, Sabrina; Leongito, Maddalena; Piccirillo, Mauro; de Angelis, Cristina; Pivonello, Claudia; Granata, Vincenza; Izzo, Francesco

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide with high morbidity, mortality and increasing incidence. It is of note that the main curative therapies for HCC are hepatic resection and transplantation although the majority of patients at the time of presentation are not eligible for resection or orthotopic liver transplantation (OLT) due to the underlying cirrhosis. Currently, a variety of loco-regional therapies, including radiofrequency ablation (RFA), percutaneous ethanol injection (PEI), microwave coagulation therapy (MCT), transarterial chemoembolization (TACE) and others, have been developed as alternative treatment options for HCC. Among these techniques, RFA is currently the most widely used treatment, due to its several advantages, such as safety and efficacy. To date, the effectiveness of RFA for HCC is reduced by the presence of residual tumor as a consequence of insufficient treatment. In order to ameliorate the effects of RFA on HCC, several in vivo studies, have been performed on its application as single or in combination treatment with drugs or others loco-regional therapies, by using rabbit VX2 liver model. This represents an ideal model of liver cancers and is widely used for imaging and other experimental studies due to the rapid growth of these tumors and their similarity to human hepatocellular carcinoma. In order to elucidate the therapeutic potential of RFA with adjuvant treatments for HCC, we reviewed the latest findings on the RFA-based studies in rabbit VX2 hepatocarcinoma models. PMID:27525037

  19. Autoregulatory loop of nuclear corepressor 1 expression controls invasion, tumor growth, and metastasis.

    PubMed

    Martínez-Iglesias, Olaia A; Alonso-Merino, Elvira; Gómez-Rey, Sara; Velasco-Martín, Juan Pedro; Martín Orozco, Rosa; Luengo, Enrique; García Martín, Rosa; Ibáñez de Cáceres, Inmaculada; Fernández, Agustín F; Fraga, Mario F; González-Peramato, Pilar; Varona, Constantino; Palacios, José; Regadera, Javier; Aranda, Ana

    2016-01-19

    Nuclear corepressor 1 (NCoR) associates with nuclear receptors and other transcription factors leading to transcriptional repression. We show here that NCoR depletion enhances cancer cell invasion and increases tumor growth and metastatic potential in nude mice. These changes are related to repressed transcription of genes associated with increased metastasis and poor prognosis in patients. Strikingly, transient NCoR silencing leads to heterochromatinization and stable silencing of the NCoR gene, suggesting that NCoR loss can be propagated, contributing to tumor progression even in the absence of NCoR gene mutations. Down-regulation of the thyroid hormone receptor β1 (TRβ) appears to be associated with cancer onset and progression. We found that expression of TRβ increases NCoR levels and that this induction is essential in mediating inhibition of tumor growth and metastasis by this receptor. Moreover, NCoR is down-regulated in human hepatocarcinomas and in the more aggressive breast cancer tumors, and its expression correlates positively with that of TRβ. These data provide a molecular basis for the anticancer actions of this corepressor and identify NCoR as a potential molecular target for development of novel cancer therapies. PMID:26729869

  20. Autoregulatory loop of nuclear corepressor 1 expression controls invasion, tumor growth, and metastasis

    PubMed Central

    Martínez-Iglesias, Olaia A.; Alonso-Merino, Elvira; Gómez-Rey, Sara; Velasco-Martín, Juan Pedro; Martín Orozco, Rosa; Luengo, Enrique; García Martín, Rosa; Ibáñez de Cáceres, Inmaculada; Fernández, Agustín F.; Fraga, Mario F.; González-Peramato, Pilar; Varona, Constantino; Palacios, José; Regadera, Javier; Aranda, Ana

    2016-01-01

    Nuclear corepressor 1 (NCoR) associates with nuclear receptors and other transcription factors leading to transcriptional repression. We show here that NCoR depletion enhances cancer cell invasion and increases tumor growth and metastatic potential in nude mice. These changes are related to repressed transcription of genes associated with increased metastasis and poor prognosis in patients. Strikingly, transient NCoR silencing leads to heterochromatinization and stable silencing of the NCoR gene, suggesting that NCoR loss can be propagated, contributing to tumor progression even in the absence of NCoR gene mutations. Down-regulation of the thyroid hormone receptor β1 (TRβ) appears to be associated with cancer onset and progression. We found that expression of TRβ increases NCoR levels and that this induction is essential in mediating inhibition of tumor growth and metastasis by this receptor. Moreover, NCoR is down-regulated in human hepatocarcinomas and in the more aggressive breast cancer tumors, and its expression correlates positively with that of TRβ. These data provide a molecular basis for the anticancer actions of this corepressor and identify NCoR as a potential molecular target for development of novel cancer therapies. PMID:26729869

  1. Evaluation of dendrimer type bio-reducible polymer as a siRNA delivery carrier for cancer therapy

    PubMed Central

    Nam, Joung-Pyo; Nam, Kihoon; Jung, Simhyun; Nah, Jae-Woon; Kim, Sung Wan

    2015-01-01

    Small interfering ribonucleic acid (siRNA), 20 – 25 base pairs in length, can interfere with the expression of specific genes. Recently, many groups reported the therapeutic intervention of siRNA in various cancer cells. In this study, dendrimer type bio-reducible polymer (PAM-ABP) which was synthesized using arginine grafted bio-reducible poly(cystaminebisacrylamide-diaminohexane) (ABP) and polyamidoamine (PAMAM) was used to deliver anti-VEGF siRNA into cancer cell lines including human hepatocarcinoma (Huh-7), human lung adenocarcinoma (A549), and human fibrosarcoma (HT1080) cells and access their potential as a siRNA delivery carrier for cancer therapy. PAM-ABP and siRNA formed polyplexes with average diameter of 116 nm and charge of around +24.6 mV. The siRNA in the PAM-ABP/siRNA polyplex released by 5 mM DTT and heparin. VEGF gene silencing efficiency of PAM-ABP/siRNA polyplexes was shown to be more effective than PEI/siRNA polyplexes in three cell lines with the following order HT1080>A549>Huh-7. PMID:25937533

  2. Complex regulation of nucleoside transporter expression in epithelial and immune system cells.

    PubMed

    Pastor-Anglada, M; Casado, F J; Valdés, R; Mata, J; García-Manteiga, J; Molina, M

    2001-01-01

    Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells co-express several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology. PMID:11396615

  3. Effects of cytokines on CYP3A4 expression and reversal of the effects by anti-cytokine agents in the three-dimensionally cultured human hepatoma cell line FLC-4.

    PubMed

    Mimura, Hanaka; Kobayashi, Kaoru; Xu, Linxiaoqing; Hashimoto, Mari; Ejiri, Yoko; Hosoda, Masaya; Chiba, Kan

    2015-02-01

    The expression of hepatic cytochrome P450 (CYP) enzymes is altered under pathological conditions with increased levels of cytokines. In this study, we analyzed the effects of cytokines (interleukin [IL]-1β, IL-6 and tumor necrosis factor α) on the expression of CYP3A4 using newly introduced three-dimensionally cultured human hepatocarcinoma FLC-4 cells. The mRNA level of CYP3A4 was significantly decreased by IL-1β, IL-6 and tumor necrosis factor-α. Formation of α-hydroxytriazolam catalyzed by CYP3A was decreased by IL-1β and IL-6. Pre-treatment with IL-6 enhanced the cytotoxic effects of gefitinib and paclitaxel. In addition, tocilizumab and IL-1 receptor antagonist restored the decreased expression of CYP3A4 mRNA by IL-6 and IL-1β, respectively. These results obtained by using three-dimensionally cultured FLC-4 cells are consistent with results obtained by using primary human hepatocytes and results of clinical studies. Therefore, three-dimensionally cultured FLC-4 cell system may be a promising cellular tool to assess the effects of cytokines on CYP3A4 expression. PMID:25760537

  4. N-Phenyl-2-p-tolylthiazole-4-carboxamide derivatives: Synthesis and cytotoxicity evaluation as anticancer agents

    PubMed Central

    Mohammadi-Farani, Ahmad; Foroumadi, Alireza; Kashani, Monireh Rezvani; Aliabadi, Alireza

    2014-01-01

    Objective(s): According to the prevalence of neoplastic diseases, there is a deep necessity for discovery of novel anticancer drugs in the field of medicinal chemistry. In the current study, a new series of phenylthiazole derivatives (compounds 4a-4f) was synthesized and their anticancer activity was assessed in vitro. Materials and Methods: All synthesized derivatives were evaluated towards three human cancerous cell lines of SKNMC (Neuroblastoma), Hep-G2 (Human hepatocarcinoma) and MCF-7 cell (Breast cancer) using MTT assay and obtained values (IC50 ± SD) were compared with doxorubicin. Results: Unfortunately, none of the synthesized compounds showed superior activity than doxorubicin against cancerous cell lines. MCF-7 cell line was the most resistant cell line against tested compounds. Compounds 4c with para nitro (IC50 = 10.8 ± 0.08 µM) and 4d with meta chlorine (IC50 = 11.6 ± 0.12 µM) moieties exerted the highest cytotoxic effects towards SKNMC and Hep-G2 cell lines respectively. Conclusion: A new series of phenylthiazole derivatives were synthesized and their anticancer activity was assessed against cancerous cell lines. More structural modifications and derivatization is necessary to achieve to the more potent compounds. PMID:25429341

  5. New 15-membered tetraaza (N4) macrocyclic ligand and its transition metal complexes: Spectral, magnetic, thermal and anticancer activity

    NASA Astrophysics Data System (ADS)

    El-Boraey, Hanaa A.; EL-Gammal, Ohyla A.

    2015-03-01

    Novel tetraamidemacrocyclic 15-membered ligand [L] i.e. naphthyl-dibenzo[1,5,9,12]tetraazacyclopentadecine-6,10,11,15-tetraoneand its transition metal complexes with Fe(II), Co(II), Ni(II), Cu(II), Ru(III) and Pd(II) have been synthesized and characterized by elemental analysis, spectral, thermal as well as magnetic and molar conductivity measurements. On the basis of analytical, spectral (IR, MS, UV-Vis, 1H NMR and EPR) and thermal studies distorted octahedral or square planar geometry has been proposed for the complexes. The antitumor activity of the synthesized ligand and some complexes against human breast cancer cell lines (MCF-7) and human hepatocarcinoma cell lines (HepG2) has been studied. The complexes (IC50 = 2.27-2.7, 8.33-31.1 μg/mL, respectively) showed potent antitumor activity, towards the former cell lines comparable with their ligand (IC50 = 13, 26 μg/mL, respectively). The results show that the activity of the ligand towards breast cancer cell line becomes more pronounced and significant when coordinated to the metal ion.

  6. Peroxisome induction potential and lipid-regulating activity in rats. Quantitative microscopy and chemical structure-activity relationships.

    PubMed Central

    McGuire, E. J.; Lucas, J. A.; Gray, R. H.; de la Iglesia, F. A.

    1991-01-01

    Structurally diverse lipid-regulating agents induce hepatomegaly, hepatic peroxisome proliferation, and hepatocarcinoma in rats by mechanisms not fully understood. Nevertheless the initial hepatic response is a prompt, florid proliferation of peroxisomes. In investigations reported here, changes in the rat hepatic peroxisome compartment were measured by quantitative microscopy to determine chemical structure requirements that relate to peroxisome proliferation and lipid regulation. Aryloxyalkanoic acids plus amide analogs, and thio, benzimidazole, phenylpiperazine, and oxazole derivatives induced peroxisome proliferation and generally decreased plasma triglyceride and total cholesterol levels. These compounds contain an acidic function or are readily metabolized to a chemical with an acidic function. Substitution of the acidic function with an adamantyloxy eliminated peroxisome proliferation and induced contrasting effects on lipid profile, increasing triglycerides and decreasing total cholesterol. A previously unreported, direct correlation emerged between peroxisome proliferation and plasma high-density lipoprotein-cholesterol levels. These effects could not be elicited separately, negating identification of functional groups that could be associated with either activity. Chemical structure and resulting peroxisome proliferation with changes in plasma lipoproteins are therefore closely interrelated in rats. Images Figure 1 PMID:1853935

  7. Sechium edule Shoot Extracts and Active Components Improve Obesity and a Fatty Liver That Involved Reducing Hepatic Lipogenesis and Adipogenesis in High-Fat-Diet-Fed Rats.

    PubMed

    Yang, Mon-Yuan; Chan, Kuei-Chuan; Lee, Yi-Ju; Chang, Xiao-Zong; Wu, Cheng-Hsun; Wang, Chau-Jong

    2015-05-13

    Excess fat accumulation in the liver increases the risk of developing progressive liver injuries ranging from a fatty liver to hepatocarcinoma. In a previous study, we demonstrated that the polyphenol components of Sechium edule shoots attenuated hepatic lipid accumulation in vitro. Therefore, we investigated the effects and mechanisms of the extract of S. edule shoots (SWE) to modulate fat accumulation in a high-fat-diet (HFD)-induced animal model. In this study, we found that the SWE can reduce the body weight, adipose tissue fat, and regulate hepatic lipid contents (e.g., triglyceride and cholesterol). Additionally, treatment of caffeic acid (CA) and hesperetin (HPT), the main ingredients of SWE, also inhibited oleic acid (OA)-induced lipid accumulation in HepG2 cells. SWE enhanced the activation of AMP-activating protein kinase (AMPK) and decreased numerous lipogenic-related enzymes, such as sterol regulator element-binding proteins (SREBPs), e.g., SREBP-1 and SREBP-2, and HMG-CoA reductase (HMGCoR) proteins, which are critical regulators of hepatic lipid metabolism. Taken together, the results demonstrated that SWE can prevent a fatty liver and attenuate adipose tissue fat by inhibiting lipogenic enzymes and stimulating lipolysis via upregulating AMPK. It was also demonstrated that the main activation components of SWE are both CA and HPT. PMID:25912298

  8. Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    PubMed Central

    Cazanave, S C; Wang, X; Zhou, H; Rahmani, M; Grant, S; Durrant, D E; Klaassen, C D; Yamamoto, M; Sanyal, A J

    2014-01-01

    Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis. PMID:24769730

  9. New gene therapy strategies for hepatic fibrosis

    PubMed Central

    Salazar-Montes, Adriana M; Hernández-Ortega, Luis D; Lucano-Landeros, Martha S; Armendariz-Borunda, Juan

    2015-01-01

    The liver is the largest internal organ of the body, which may suffer acute or chronic injury induced by many factors, leading to cirrhosis and hepatocarcinoma. Cirrhosis is the irreversible end result of fibrous scarring and hepatocellular regeneration, characterized by diffuse disorganization of the normal hepatic structure, regenerative nodules and fibrotic tissue. Cirrhosis is associated with a high co-morbidity and mortality without effective treatment, and much research has been aimed at developing new therapeutic strategies to guarantee recovery. Liver-based gene therapy has been used to downregulate specific genes, to block the expression of deleterious genes, to delivery therapeutic genes, to prevent allograft rejection and to augment liver regeneration. Viral and non-viral vectors have been used, with viral vectors proving to be more efficient. This review provides an overview of the main strategies used in liver-gene therapy represented by non-viral vectors, viral vectors, novel administration methods like hydrodynamic injection, hybrids of two viral vectors and blocking molecules, with the hope of translating findings from the laboratory to the patient´s bed-side. PMID:25852266

  10. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    SciTech Connect

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye; Hong, Darong; Jung, Bom; Park, Min-Ju; Kim, Jong-Ho

    2015-08-07

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer.

  11. Beneficial Effect of Synbiotic Supplementation on Hepatic Steatosis and Anthropometric Parameters, But Not on Gut Permeability in a Population with Nonalcoholic Steatohepatitis

    PubMed Central

    Ferolla, Silvia M.; Couto, Cláudia A.; Costa-Silva, Luciana; Armiliato, Geyza N. A.; Pereira, Cristiano A. S.; Martins, Flaviano S.; Ferrari, Maria de Lourdes A.; Vilela, Eduardo G.; Torres, Henrique O. G.; Cunha, Aloísio S.; Ferrari, Teresa C. A.

    2016-01-01

    Nonalcoholic fatty liver disease is the most prevalent chronic liver disease in Western countries; it can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocarcinoma. The importance of gut-liver-adipose tissue axis has become evident and treatments targeting gut microbiota may improve inflammatory and metabolic parameters in NASH patients. In a randomized, controlled clinical trial, involving 50 biopsy-proven NASH patients, we investigated the effects of synbiotic supplementation on metabolic parameters, hepatic steatosis, intestinal permeability, small intestinal bacterial overgrowth (SIBO) and lipopolysaccharide (LPS) serum levels. Patients were separated into two groups receiving Lactobacillus reuteri with guar gum and inulin for three months and healthy balanced nutritional counseling versus nutritional counseling alone. Before and after the intervention we assessed steatosis by magnetic resonance imaging, intestinal permeability by lactulose/mannitol urinary excretion and SIBO by glucose breath testing. NASH patients presented high gut permeability, but low prevalence of SIBO. After the intervention, only the synbiotic group presented a reduction in steatosis, lost weight, diminished BMI and waist circumference measurement. Synbiotic did not improve intestinal permeability or LPS levels. We concluded that synbiotic supplementation associated with nutritional counseling seems superior to nutritional counseling alone for NASH treatment as it attenuates steatosis and may help to achieve weight loss. PMID:27367724

  12. Antioxidant and Proapoptotic Activities of Sclerocarya birrea [(A. Rich.) Hochst.] Methanolic Root Extract on the Hepatocellular Carcinoma Cell Line HepG2

    PubMed Central

    Armentano, Maria Francesca; Bisaccia, Faustino; Miglionico, Rocchina; Russo, Daniela; Nolfi, Nicoletta; Carmosino, Monica; Andrade, Paula B.; Valentão, Patrícia; Diop, Moussoukhoye Sissokho

    2015-01-01

    The main goal of this study was to characterize the in vitro antioxidant activity and the apoptotic potential of S. birrea methanolic root extract (MRE). Among four tested extracts, obtained with different solvents, MRE showed the highest content of polyphenols, flavonoids, and tannins together with antioxidant activities tested with superoxide, nitric oxide, ABTS, and beta-carotene bleaching assays. Moreover, the cytotoxic effect of MRE was evaluated on the hepatocarcinoma cell line HepG2. In these cells, MRE treatment induced apoptosis and generated reactive oxygen species (ROS) in dose-dependent manner. The cytotoxic effect promoted by MRE was prevented by pretreatment of HepG2 cells with N-acetyl-L-cysteine (NAC), suggesting that oxidative stress was pivotal in MRE-mediated cell death. Moreover, we showed that the MRE treatment induced the mitochondrial membrane depolarization and the cytochrome c release from mitochondria into the cytosol. It suggests that the apoptosis occurred in a mitochondrial-dependent pathway. Interestingly, MRE showed a sensibly lower cytotoxicity, associated with a low increase of ROS, in normal human dermal fibroblasts compared to HepG2 cells. It is suggested that the methanolic root extract of S. Birrea is able to selectively increase intracellular ROS levels in cancer cells, promoting cell death. PMID:26075245

  13. HFE genotyping in patients with elevated serum iron indices and liver diseases.

    PubMed

    Evangelista, Andreia Silva; Nakhle, Maria Cristina; de Araújo, Thiago Ferreira; Abrantes-Lemos, Clarice Pires; Deguti, Marta Mitiko; Carrilho, Flair José; Cançado, Eduardo Luiz Rachid

    2015-01-01

    Iron abnormalities in chronic liver disease may be the result of genetic diseases or secondary factors. The present study aimed to identify subjects with HFE-HH in order to describe the frequency of clinical manifestations, identify risk factors for iron elevation, and compare the iron profile of HFE-HH to other genotypes in liver disease patients. A total of 108 individuals with hepatic disease, transferrin saturation (TS) > 45%, and serum ferritin (SF) > 350 ng/mL were tested for HFE mutations. Two groups were characterized: C282Y/C282Y or C282Y/H63D genotypes (n = 16) were the HFE hereditary hemochromatosis (HFE-HH) group; and C282Y and H63D single heterozygotes, the H63D/H63D genotype, and wild-type were considered group 2 (n = 92). Nonalcoholic liver disease, alcoholism, and chronic hepatitis C were detected more frequently in group 2, whereas arthropathy, hepatocarcinoma, diabetes, and osteoporosis rates were significantly higher in the HFE-HH group. TS > 82%, SF > 2685 ng/mL, and serum iron > 178 μg/dL were the cutoffs for diagnosis of HFE-HH in patients with liver disease. Thus, in non-Caucasian populations with chronic liver disease, HFE-HH diagnosis is more predictable in those with iron levels higher than those proposed in current guidelines for the general population. PMID:25654085

  14. Inhibitory effect of plant-originated glycoprotein (27 kDa) on expression of matrix metalloproteinase-9 in cadmium chloride-induced BNL CL.2 cells.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2011-12-01

    Cadmium is very harmful to the environment and to human beings because of its long lifetime. The toxicity of cadmium as an industrial pollutant and a food contaminant, and as one of the major components in cigarette smoke is well known. Cadmium can cause a number of lesions in many organs, such as the kidney, the lung, the liver, the brain, the blood system. However, the mechanism of toxicity of cadmium is not yet clear. Also, it has been well known as human carcinogen which is indirectly caused inflammation-mediated hepatocarcinoma. In the present study it was demonstrated that glycoprotein (27 kDa) isolated from Gardenia jasminoides Ellis (GJE) protects BNL CL.2 cells from expression of inflammation-related factors stimulated by cadmium chloride (10 μM). Intracellular ROS and intracellular Ca(2+) using fluorescence, activities of activator protein (AP)-1, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, and arachidonic acid (AA) using immunoblot analysis or radioactivity were evaluated. The results obtained from this experiment indicated that GJE glycoprotein (100 μg/mL) inhibits the production of intracellular ROS, and intracellular Ca(2+) mobilization. Also, it significantly suppressed inflammatory factors [expression of AP-1 (c-Jun and c-Fos), arachidonic acid, COX-2, and MMP-9]. Taken together, these findings suggest that GJE glycoprotein might be used for protection of inflammation caused by cadmium ion as one of natural compounds. PMID:21924884

  15. [Management and treatment of patients with hepatitis B].

    PubMed

    den Eynde, Eva Van; Riveiro-Barciela, Mar

    2016-07-01

    Chronic hepatitis B is a major cause of morbidity and mortality worldwide. Approximately one third of the world's population has serological evidence of past or present infection by hepatitis B virus (HBV) and 350-400 million people are chronic HBV surface antigen carriers. The aim of therapy is to prevent the onset of liver fibrosis and development of cirrhosis or hepatocarcinoma by sustained suppression of viral replication. Currently there are 2 strategies for the treatment of chronic hepatitis B: the pegylated interferon and long-term treatment with nucleoside/nucleotide analogues. Pegylated interferon has the advantage of being a treatment of limited duration, and is particularly suitable for patients with chronic hepatitis with positive HBeAg (hepatitis B e antigen), but the unfavorable adverse event profile and route of parenteral administration makes it less used than nucleoside/nucleotide analogues. Tenofovir and entecavir have shown to be potent inhibitors of HBV with a high genetic barrier to resistance and few adverse effects, so are considered as the first line therapy. PMID:27474247

  16. Coordination behavior of tetraaza [N4] ligand towards Co(II), Ni(II), Cu(II), Cu(I) and Pd(II) complexes: Synthesis, spectroscopic characterization and anticancer activity

    NASA Astrophysics Data System (ADS)

    El-Boraey, Hanaa A.

    2012-11-01

    Novel eight Co(II), Ni(II), Cu(II), Cu(I) and Pd(II) complexes with [N4] ligand (L) i.e. 2-amino-N-{2-[(2-aminobenzoyl)amino]ethyl}benzamide have been synthesized and structurally characterized by elemental analysis, spectral, thermal (TG/DTG), magnetic, and molar conductivity measurements. On the basis of IR, mass, electronic and EPR spectral studies an octahedral geometry has been proposed for Co(II), Ni(II) complexes and Cu(II) chloride complex, square-pyramidal for Cu(I) bromide complex. For Cu(II) nitrate complex (6), Pd(II) complex (8) square planar geometry was proposed. The EPR data of Cu(II) complexes in powdered form indicate dx2-y2 ground state of Cu(II) ion. The antitumor activity of the synthesized ligand and some selected metal complexes has been studied. The palladium(II) complex (8) was found to display cytotoxicity (IC50 = 25.6 and 41 μM) against human breast cancer cell line MCF-7 and human hepatocarcinoma HEPG2 cell line.

  17. Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX

    PubMed Central

    Zhang, Liqin; Delgado, Stefanie; Champanhac, Carole; Cansiz, Sena; Wu, Cuichen; Shan, Hong; Tan, Weihong

    2015-01-01

    Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment), have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers. PMID:25938802

  18. N-acetylcysteine improves antitumoural response of Interferon alpha by NF-kB downregulation in liver cancer cells

    PubMed Central

    2012-01-01

    Background Liver cancer is one of the most common malignancies in the world and at the moment, there is no drug intervention effective for the treatment of liver tumours. Investigate the effect of N-acetylcysteine (NAC), which has been studied for its antitumoural properties, on the toxicity of hepatocarcinoma (HCC) cells in vitro when used with the drug interferon alpha-2A (IFN), which is used clinically to treat HCC. Results NAC, IFN and NAC plus IFN reduced cell viability, as determined by MTT assay. More importantly, NAC potentiates the cytotoxic effect of IFN, with the best response achieved with 10 mM of NAC and 2.5 x 104 of IFN. These results were confirmed by Annexin/PI staining through flow cytometry and morphologic analyses. Co-treatment reduced the expression of the nuclear transcription factor kappa-B (NF-kB). In a similar way to NAC, RNAi against p65 potentiated the toxic effect of IFN, suggesting that, indeed, NAC may be enhancing the effect of IFN through inhibition of NF-kB. Conclusions Our results support the notion that NAC may be an important drug for the treatment of liver tumours as primary or adjuvant therapy. IFN has a limited clinical response, and therefore, the anti-proliferative properties of NAC in the liver should be explored further as an alternative for non-responders to IFN treatment. PMID:23206959

  19. Antioxidant and Antitumor Activities of the Extracts from Chinese Yam (Dioscorea opposite Thunb.) Flesh and Peel and the Effective Compounds.

    PubMed

    Liu, Yuanxue; Li, Hongfa; Fan, Yaya; Man, Shuli; Liu, Zhen; Gao, Wenyuan; Wang, Tingting

    2016-06-01

    The aims of this study are to investigate the antioxidant and antitumor activities of the water and ethanol extracts isolated from Chinese yam (Dioscorea opposite Thunb.) flesh (CYF) and peel (CYP) and the effective compounds. It was found that all peel portions have a better effect on reactive oxygen (ROS) scavenging assay than meat portions, especially for the water extract of Chinese yam peel (CYP-W). Its IC50 values for hydroxyl radical (OH•) scavenging assay (744.25 ± 3.46 μg/mL) and for 1,1-diphenyl-2-picrylhydrazyl scavenging assay (374.85 ± 6.78 μg/mL) were both lower than that of yam flesh (CYF-W). Furthermore, the antitumor property of yam peel was more effective than that of yam flesh (CYF-W) on mouse models, with tumor inhibition rates were 47.92% and 27.41% for Ehrlich Ascites Tumor (EAC) model and 40.44% and 24.22% for H22 hepatocarcinoma tumor (H22) model. Meanwhile, extracts of peel showed higher allantoin, total flavonoids, and total phenolics contents than extracts of flesh. In conclusion, this study demonstrated that CYP-W exerted better antitumor activity than flesh extracts and the scavenging ROS effects were also significantly higher in the CYP-W in vitro. Moreover, the data indicated that allantoin may play an important role on antioxidative and antitumor capacity in yam peel. PMID:27122252

  20. A three dimensional anchorage independent in vitro system for the prolonged growth of embryoid bodies to study cancer cell behaviour and anticancer agents.

    PubMed

    Fong, Chui-Yee; Chak, Li-Ling; Subramanian, Arjunan; Tan, Jee-Hian; Biswas, Arijit; Gauthaman, Kalamegam; Choolani, Mahesh; Chan, Woon-Khiong; Bongso, Ariff

    2009-12-01

    We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior. PMID:20058203

  1. Behavioral characteristics of a mouse model of cancer pain.

    PubMed

    Lee, Bae Hwan; Seong, Jinsil; Kim, Un Jeng; Won, Ran; Kim, Jiyoung

    2005-04-30

    Pain is a major symptom in cancer patients, and most cancer patients with advanced or terminal cancers suffer from chronic pain related to treatment failure and/or tumor progression. In the present study, we examined the development of cancer pain in mice. Murine hepatocarcinoma cells, HCa-1, were inoculated unilaterally into the thigh or the dorsum of the foot of male C3H/HeJ mice. Four weeks after inoculation, behavioral signs were observed for mechanical allodynia, cold allodynia, and hyperalgesia using a von Frey filament, acetone, and radiant heat, respectively. Bone invasion by the tumor commenced from 7 days after inoculation of tumor cells and was evident from 14 days after inoculation. Cold allodynia but neither mechanical allodynia nor hyperalgesia was observed in mice that received an inoculation into the thigh. On the contrary, mechanical allodynia and cold allodynia, but not hyperalgesia, were developed in mice with an inoculation into the foot. Sometimes, mirror-image pain was developed in these animals. These results suggest that carcinoma cells injected into the foot of mice may develop severe chronic pain related to cancer. This animal model of pain would be useful to elucidate the mechanisms of cancer pain in humans. PMID:15861499

  2. Erianin induces G2/M-phase arrest, apoptosis, and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells in vitro and in vivo.

    PubMed

    Wang, H; Zhang, T; Sun, W; Wang, Z; Zuo, D; Zhou, Z; Li, S; Xu, J; Yin, F; Hua, Y; Cai, Z

    2016-01-01

    Erianin, a natural product derived from Dendrobium chrysotoxum, has exhibited potential antitumor activity in various malignancies, including hepatocarcinoma, melanoma, and promyelocytic leukemia. Here we explored the effects of erianin on osteosarcoma (OS) in vitro and in vivo and further elucidated the underlying molecule mechanisms. In this study, we found that erianin potently suppressed cell viability in various OS cell lines. Treatment with erianin induced G2/M-phase arrest, apoptosis, and autophagy in OS cells. Further studies showed that erianin-induced apoptosis and autophagy was attributed to reactive oxygen species (ROS), as N-acetyl cysteine (NAC), an ROS scavenger, attenuated them. Moreover, we found that erianin induced activation of c-Jun N-terminal kinase (JNK) signal pathway, which was also blocked by NAC. Downregulation of JNK by its specific inhibitor SP600125 could attenuate apoptosis and autophagy induced by erianin. Finally, erianin in vivo markedly reduced the growth with little organ-related toxicity. In conclusion, erianin induced cell cycle G2/M-phase arrest, apoptosis, and autophagy via the ROS/JNK signaling pathway in human OS. In light of these results, erianin may be a promising agent for anticancer therapy against OS. PMID:27253411

  3. Clusianone, a naturally occurring nemorosone regioisomer, uncouples rat liver mitochondria and induces HepG2 cell death.

    PubMed

    Reis, Felippe H Z; Pardo-Andreu, Gilberto L; Nuñez-Figueredo, Yanier; Cuesta-Rubio, Osmany; Marín-Prida, Javier; Uyemura, Sérgio A; Curti, Carlos; Alberici, Luciane C

    2014-04-01

    Clusianone is a member of the polycyclic polyprenylated acylphloroglucinol family of natural products; its cytotoxic mechanism is unknown. Clusianone is a structural isomer of nemorosone, which is a mitochondrial uncoupler and a well-known cytotoxic anti-cancer agent; thus, we addressed clusianone action at the mitochondria and its potential cytotoxic effects on cancer cells. In the HepG2 hepatocarcinoma cell line, clusianone induced mitochondrial membrane potential dissipation, ATP depletion and phosphatidyl serine externalization; this later event is indicative of apoptosis induction. In isolated mitochondria from rat liver, clusianone promoted protonophoric mitochondrial uncoupling. This was evidenced by the dissipation of mitochondrial membrane potential, an increase in resting respiration, an inhibition of Ca(2+) influx, stimulation of Ca(2+) efflux in Ca(2+)-loaded mitochondria, a decrease in ATP and NAD(P)H levels, generation of ROS, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. The cytotoxic and uncoupling actions of clusianone were appreciably less than those of nemorosone, likely due to the presence of an intra-molecular hydrogen bond with the juxtaposed carbonyl group at the C15 position. Therefore, clusianone is capable of pharmacologically increasing the leakage of protons from the mitochondria and with favorable cytotoxicity in relation to nemorosone. PMID:24491676

  4. Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

    PubMed Central

    Spencer, John David; Jackson, Ashley R.; Li, Birong; Ching, Christina B.; Vonau, Martin; Easterling, Robert S.; Schwaderer, Andrew L.; McHugh, Kirk M.; Becknell, Brian

    2015-01-01

    Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain. PMID:26658437

  5. Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

    PubMed Central

    Storm, Michael P.; Sorrell, Ian; Shipley, Rebecca; Regan, Sophie; Luetchford, Kim A.; Sathish, Jean; Webb, Steven; Ellis, Marianne J.

    2016-01-01

    Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture. PMID:27285826

  6. Dose-dependent cytotoxic effects of boldine in HepG-2 cells-telomerase inhibition and apoptosis induction.

    PubMed

    Noureini, Sakineh Kazemi; Wink, Michael

    2015-01-01

    Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine) exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT) gene (p < 0.01) and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002). However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02) and p21 (p < 0.01) genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations. PMID:25719742

  7. Huaier Aqueous Extract Induces Hepatocellular Carcinoma Cells Arrest in S Phase via JNK Signaling Pathway

    PubMed Central

    Zhang, Chengshuo; Zhang, Jialin; Li, Xin; Sun, Ning; Yu, Rui; Zhao, Bochao; Yu, Dongyang; Cheng, Ying; Liu, Yongfeng

    2015-01-01

    Huaier aqueous extract, the main active constituent of Huaier proteoglycan, has antihepatocarcinoma activity in experimental and clinical settings. However, the potential and associated antihepatoma mechanisms of Huaier extract are not yet fully understood. Therefore, in this study, we aimed to elucidate the inhibitory proliferation effect of Huaier extract on apoptosis and cycle of HepG2 and Bel-7402 cells. Our data demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability dose-dependently. Flow cytometric analysis showed that a 48 h treatment of Huaier extract caused cell apoptosis. Typical apoptotic nucleus alterations were observed with fluorescence microscope after Hoechst staining. Immunoblot analysis further demonstrated that Huaier extract activated caspase 3 and PARP. Additionally, Huaier extract inhibited the activity of p-ERK, p-p38, and p-JNK in terms of MAPK. Furthermore, Huaier extract induced HCC cells arrest in S phase and decreased the cycle related protein expression of β-catenin and cyclin D1. Studies with JNK specific inhibitor, SP600125, showed that Huaier extract induced S phase arrest and decreased β-catenin and cyclin D1 expression via JNK signaling pathway. In conclusion, we verify that Huaier extract causes cell apoptosis and induces hepatocellular carcinoma cells arrest in S phase via JNK pathway, which advances our understanding on the molecular mechanisms of Huaier extract in hepatocarcinoma management. PMID:26229542

  8. Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

    PubMed

    Shigeto, Hajime; Nakatsuka, Keisuke; Ikeda, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Funabashi, Hisakage

    2016-08-16

    This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner. PMID:27458920

  9. Constitutive hepcidin expression prevents iron overload in a mouse model of hemochromatosis.

    PubMed

    Nicolas, Gaël; Viatte, Lydie; Lou, Dan-Qing; Bennoun, Myriam; Beaumont, Carole; Kahn, Axel; Andrews, Nancy C; Vaulont, Sophie

    2003-05-01

    Hereditary hemochromatosis is a prevalent genetic disorder of iron hyperabsorption leading to hyperferremia, tissue iron deposition and complications including cirrhosis, hepatocarcinoma, cardiomyopathy and diabetes. Most individuals affected with hereditary hemochromatosis are homozygous with respect to a missense mutation that disrupts the conformation of HFE, an atypical HLA class I molecule (ref. 1; OMIM 235200). Mice lacking Hfe or producing a C282Y mutant Hfe protein develop hyperferremia and have high hepatic iron levels. In both humans and mice, hereditary hemochromatosis is associated with a paucity of iron in reticuloendothelial cells. It has been suggested that HFE modulates uptake of transferrin-bound iron by undifferentiated intestinal crypt cells, thereby programming the absorptive capacity of enterocytes derived from these cells; however, this model is unproven and controversial. Hepcidin, a peptide hormone (HAMP; OMIM 606464), seems to act in the same regulatory pathway as HFE. Although expression of mouse Hamp is normally greater during iron overload, Hfe-/- mice have inappropriately low expression of Hamp. We crossed Hfe-/- mice with transgenic mice overexpressing Hamp and found that Hamp inhibited the iron accumulation normally observed in the Hfe-/- mice. This argues against the crypt programming model and suggests that failure of Hamp induction contributes to the pathogenesis of hemochromatosis, providing a rationale for the use of HAMP in the treatment of this disease. PMID:12704388

  10. The Thyroid Hormone Receptors Inhibit Hepatic Interleukin-6 Signaling During Endotoxemia

    PubMed Central

    Contreras-Jurado, Constanza; Alonso-Merino, Elvira; Saiz-Ladera, Cristina; Valiño, Arturo José; Regadera, Javier; Alemany, Susana; Aranda, Ana

    2016-01-01

    Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-κB and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections. PMID:27484112

  11. The Thyroid Hormone Receptors Inhibit Hepatic Interleukin-6 Signaling During Endotoxemia.

    PubMed

    Contreras-Jurado, Constanza; Alonso-Merino, Elvira; Saiz-Ladera, Cristina; Valiño, Arturo José; Regadera, Javier; Alemany, Susana; Aranda, Ana

    2016-01-01

    Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-κB and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections. PMID:27484112

  12. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends.

    PubMed

    Bernardino, Raquel L; Marinelli, Raul A; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G; Svelto, Maria; Oliveira, Pedro F; Calamita, Giuseppe

    2016-01-01

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field. PMID:27409609

  13. Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

    NASA Astrophysics Data System (ADS)

    Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

    2016-04-01

    Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and