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Sample records for acid induced cell

  1. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  2. High dose of ascorbic acid induces cell death in mesothelioma cells.

    PubMed

    Takemura, Yukitoshi; Satoh, Motohiko; Satoh, Kiyotoshi; Hamada, Hironobu; Sekido, Yoshitaka; Kubota, Shunichiro

    2010-04-02

    Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.

  3. Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

    PubMed

    Rao, M V; Lee, H; Creelman, R A; Mullet, J E; Davis, K R

    2000-09-01

    Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

  4. Docosahexaenoic Acid Induces Apoptosis in Primary Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Gyan, Emmanuel; Tournilhac, Olivier; Halty, Christelle; Veyrat-Masson, Richard; Akil, Saïda; Berger, Marc; Hérault, Olivier; Callanan, Mary; Bay, Jacques-Olivier

    2015-01-01

    Chronic lymphocytic leukemia is an indolent disorder with an increased infectious risk remaining one of the main causes of death. Development of therapies with higher safety profile is thus a challenging issue. Docosahexaenoic acid (DHA, 22:6) is an omega-3 fatty acid, a natural compound of normal cells, and has been shown to display antitumor potency in cancer. We evaluated the potential in vitro effect of DHA in primary CLL cells. DHA induces high level of in vitro apoptosis compared to oleic acid in a dose-dependent and time-dependent manner. Estimation of IC50 was only of 4.813 µM, which appears lower than those reported in solid cancers. DHA is highly active on CLL cells in vitro. This observation provides a rationale for further studies aiming to understand its mechanisms of action and its potent in vivo activity. PMID:26734128

  5. Gambogic acid induces apoptosis in diffuse large B-cell lymphoma cells via inducing proteasome inhibition.

    PubMed

    Shi, Xianping; Lan, Xiaoying; Chen, Xin; Zhao, Chong; Li, Xiaofen; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Zang, Dan; Liao, Yuning; Zhang, Peiquan; Wang, Xuejun; Liu, Jinbao

    2015-04-08

    Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment.

  6. Retinoic acid from retinal pigment epithelium induces T regulatory cells.

    PubMed

    Kawazoe, Yuko; Sugita, Sunao; Keino, Hiroshi; Yamada, Yukiko; Imai, Ayano; Horie, Shintaro; Mochizuki, Manabu

    2012-01-01

    Primary cultured retinal pigment epithelial (RPE) cells can convert T cells into T regulatory cells (Tregs) through inhibitory factor(s) including transforming growth factor β (TGFβ) in vitro. Retinoic acid (RA) enhances induction of CD4(+) Tregs in the presence of TGFβ. We investigated whether RA produced by RPE cells can promote generation of Tregs. We found that in vitro, RA-treated T cells expressed high levels of Foxp3 in the presence of recombinant TGFβ. In GeneChip analysis, cultured RPE cells constitutively expressed RA-associated molecules such as RA-binding proteins, enzymes, and receptors. RPE from normal mice, but not vitamin A-deficient mice, contained significant levels of TGFβ. RPE-induced Tregs from vitamin A-deficient mice failed to suppress activation of target T cells. Only a few Foxp3(+) T cells were found in intraocular cells from vitamin A-deficient experimental autoimmune uveitis (EAU) mice, whereas expression was higher in cells from normal EAU mice. RA receptor antagonist-pretreated or RA-binding protein-siRNA-transfected RPE cells failed to convert CD4(+) T cells into Tregs. Our data support the hypothesis that RPE cells produce RA, thereby enabling bystander T cells to be converted into Tregs through TGFβ promotion, which can then participate in the establishment of immune tolerance in the eye.

  7. Lysophosphatidic acid-induced chemotaxis of bone cells.

    SciTech Connect

    Karagiosis, Sue A.; Masiello, Lisa M.; Bollinger, Nikki; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a platelet-derived bioactive lipid that is postulated to regulate wound healing. LPA activates G protein-coupled receptors to induce Ca2+ signaling in MC3T3-E1 pre-osteoblasts, and is a potent chemotactic stimulus for these cells. Since bone fracture healing requires the migration of osteoblast progenitors, we postulate that LPA is among the factors that stimulate bone repair. UMR 106-01 cells, which express a more mature osteoblastic phenotype than MC3T3-E1 cells, did not migrate in response to LPA, although they express LPA receptors and exhibit LPA-induced Ca2+ signals. This suggests that LPA differentially induces pre-osteoblast chemotaxis, consistent with our hypothesis that LPA stimulates the motility of osteoblast progenitors during bone healing. LPA-stimulated MC3T3-E1 cells exhibit striking changes in morphology and F-actin architecture, and phosphatidylinositol-3 kinase (PI3K) is required for motility-associated cytoskeletal rearrangements in many cell types. We found a dose-dependent reduction in LPA-induced osteoblast migration when cells also were treated with the PI3K inhibitor, LY294002. Treatment of many cell types with LPA is associated with an autocrine/paracrine transactivation of the EGF receptor (EGFR) via shedding of surface-tethered EGFR ligands, a phenomenon often required for LPA-induced chemotaxis. MC3T3-E1 cells express multiple EGFR ligands (epigen, epiregulin, HB-EGF and amphiregulin) and migrated in response to EGF. However, while EGF-stimulated motility in MC3T3-E1 cells was blocked by an EGFR inhibitor, there was no significant effect on LPA-induced chemotaxis. Activation of MAP kinases is a hallmark of EGFR-mediated signaling, and EGF treatment of MC3T3-E1 cells led to a strong stimulation of ERK1/2 kinase. In contrast, LPA induced only a minor elevation in ERK activity. Thus, it is likely that the increase in ERK activity by LPA is related to cell proliferation associated with lipid treatment. We

  8. Monomethylarsonous acid induces transformation of human bladder cells

    SciTech Connect

    Bredfeldt, Tiffany G.; Jagadish, Bhumasamudram; Eblin, Kylee E.; Mash, Eugene A.; Gandolfi, A. Jay . E-mail: gandolfi@pharmacy.arizona.edu

    2006-10-01

    Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMA{sup III}) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 {mu}M MMA{sup III} for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMA{sup III}, which had a 40-50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMA{sup III} exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMA{sup III} for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMA{sup III}. These observations are the first demonstration of MMA{sup III}-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMA{sup III} may be carcinogenic in human tissues.

  9. Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae

    PubMed Central

    Rego, António; Duarte, Ana M.; Azevedo, Flávio; Sousa, Maria J.; Côrte-Real, Manuela; Chaves, Susana R.

    2014-01-01

    Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria. PMID:28357256

  10. Phenylpropenoic Acid Glucoside from Rooibos Protects Pancreatic Beta Cells against Cell Death Induced by Acute Injury

    PubMed Central

    Himpe, Eddy; Cunha, Daniel A.; Song, Imane; Bugliani, Marco; Marchetti, Piero; Cnop, Miriam; Bouwens, Luc

    2016-01-01

    Objective Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress. Methods Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells. Results Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E beta cells from streptozotocin-induced apoptosis and necrosis in a BCL2-dependent and independent way, respectively, depending on glucose concentration. PPAG also protected human pancreatic islet cells against the cytotoxic action of the fatty acid palmitate. Conclusions These findings show the potential use of PPAG as phytomedicine which protects the beta cell mass exposed to acute diabetogenic stress. PMID:27299564

  11. Lysophosphatidic acid induced red blood cell aggregation in vitro.

    PubMed

    Kaestner, Lars; Steffen, Patrick; Nguyen, Duc Bach; Wang, Jue; Wagner-Britz, Lisa; Jung, Achim; Wagner, Christian; Bernhardt, Ingolf

    2012-10-01

    Under physiological conditions healthy RBCs do not adhere to each other. There are indications that RBCs display an intercellular adhesion under certain (pathophysiological) conditions. Therefore we investigated signaling steps starting with transmembrane calcium transport by means of calcium imaging. We found a lysophosphatidic acid (LPA) concentration dependent calcium influx with an EC(50) of 5 μM LPA. Downstream signaling was investigated by flow cytometry as well as by video-imaging comparing LPA induced with "pure" calcium mediated phosphatidylserine exposure and concluded the coexistence of two branches of the signaling pathway. Finally we performed force measurements with holographic optical tweezers (HOT): The intercellular adhesion of RBCs (aggregation) exceeds a force of 25 pN. These results support (i) earlier data of a RBC associated component in thrombotic events under certain pathophysiological conditions and (ii) the concept to use RBCs in studies of cellular adhesion behavior, especially in combination with HOT. The latter paves the way to use RBCs as model cells to investigate molecular regulation of cellular adhesion processes.

  12. Valproic acid induces apoptosis and cell cycle arrest in poorly differentiated thyroid cancer cells.

    PubMed

    Catalano, Maria G; Fortunati, Nicoletta; Pugliese, Mariateresa; Costantino, Lucia; Poli, Roberta; Bosco, Ornella; Boccuzzi, Giuseppe

    2005-03-01

    Poorly differentiated thyroid carcinoma is an aggressive human cancer that is resistant to conventional therapy. Histone deacetylase inhibitors are a promising class of drugs, acting as antiproliferative agents by promoting differentiation, as well as inducing apoptosis and cell cycle arrest. Valproic acid (VPA), a class I selective histone deacetylase inhibitor widely used as an anticonvulsant, promotes differentiation in poorly differentiated thyroid cancer cells by inducing Na(+)/I(-) symporter and increasing iodine uptake. Here, we show that it is also highly effective at suppressing growth in poorly differentiated thyroid cancer cell lines (N-PA and BHT-101). Apoptosis induction and cell cycle arrest are the underlying mechanisms of VPA's effect on cell growth. It induces apoptosis by activating the intrinsic pathway; caspases 3 and 9 are activated but not caspase 8. Cell cycle is selectively arrested in G(1) and is associated with the increased expression of p21 and the reduced expression of cyclin A. Both apoptosis and cell cycle arrest are induced by treatment with 1 mm VPA, a dose that promotes cell redifferentiation and that is slightly above the serum concentration reached in patients treated for epilepsy. These multifaceted properties make VPA of clinical interest as a new approach to treating poorly differentiated thyroid cancer.

  13. Hepatic Stellate Cells Preferentially Induce Foxp3+ Regulatory T Cells by Production of Retinoic Acid

    PubMed Central

    Dunham, Richard M.; Thapa, Manoj; Velazquez, Victoria M.; Elrod, Elizabeth J.; Denning, Timothy L.; Pulendran, Bali

    2013-01-01

    The liver has long been described as immunosuppressive, although the mechanisms underlying this phenomenon are incompletely understood. Hepatic stellate cells (HSCs), a population of liver nonparenchymal cells, are potent producers of the regulatory T cell (Treg)–polarizing molecules TGF-β1 and all-trans retinoic acid, particularly during states of inflammation. HSCs are activated during hepatitis C virus infection and may therefore play a role in the enrichment of Tregs during infection. We hypothesized that Ag presentation in the context of HSC activation will induce naive T cells to differentiate into Foxp3+ Tregs. To test this hypothesis, we investigated the molecular interactions between murine HSCs, dendritic cells, and naive CD4+ T cells. We found that HSCs alone do not present Ag to naive CD4+ T cells, but in the presence of dendritic cells and TGF-β1, preferentially induce functional Tregs. This Treg induction was associated with retinoid metabolism by HSCs and was dependent on all-trans retinoic acid. Thus, we conclude that HSCs preferentially generate Foxp3+ Tregs and, therefore, may play a role in the tolerogenic nature of the liver. PMID:23359509

  14. Palmitic acid but not palmitoleic acid induces insulin resistance in a human endothelial cell line by decreasing SERCA pump expression.

    PubMed

    Gustavo Vazquez-Jimenez, J; Chavez-Reyes, Jesus; Romero-Garcia, Tatiana; Zarain-Herzberg, Angel; Valdes-Flores, Jesus; Manuel Galindo-Rosales, J; Rueda, Angelica; Guerrero-Hernandez, Agustin; Olivares-Reyes, J Alberto

    2016-01-01

    Palmitic acid is a negative regulator of insulin activity. At the molecular level, palmitic acid reduces insulin stimulated Akt Ser473 phosphorylation. Interestingly, we have found that incubation with palmitic acid of human umbilical vein endothelial cells induced a biphasic effect, an initial transient elevation followed by a sustained reduction of SERCA pump protein levels. However, palmitic acid produced a sustained inhibition of SERCA pump ATPase activity. Insulin resistance state appeared before there was a significant reduction of SERCA2 expression. The mechanism by which palmitic acid impairs insulin signaling may involve endoplasmic reticulum stress, because this fatty acid induced activation of both PERK, an ER stress marker, and JNK, a kinase associated with insulin resistance. None of these effects were observed by incubating HUVEC-CS cells with palmitoleic acid. Importantly, SERCA2 overexpression decreased the palmitic acid-induced insulin resistance state. All these results suggest that SERCA pump might be the target of palmitic acid to induce the insulin resistance state in a human vascular endothelial cell line. Importantly, these data suggest that HUVEC-CS cells respond to palmitic acid-exposure with a compensatory overexpression of SERCA pump within the first hour, which eventually fades out and insulin resistance prevails.

  15. Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kurita-Ochiai, Tomoko; Fukushima, Kazuo; Ochiai, Kuniyasu

    1999-01-01

    We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+ and CD8+ cells. PMID:9864191

  16. Dimethylarsenic acid induces tetraploids in Chinese hamster cells

    SciTech Connect

    Endo, Ginji; Horiguchi, Shun'ichi ); Kuroda, Koichi; Okamoto, Akiyoshi )

    1992-01-01

    Arsenic has been documented as a human carcinogen of the skin and lungs. However, attempts to induce tumors in experimental animals with inorganoarsenic compounds have mostly failed except in a few studies in which animals were given arsenic trioxide by intratracheal instillation. Moreover, inorganoarsenics are either inactive or too weak to induce gene mutations in vitro. The mechanism of arsenic carcinogenicity has not yet been discovered. Most mammals including human are able to methylate inorganoarsenic compounds to methylarsonic acid and dimethylarsenic acid. However, the genotoxicity of organoarsenic compounds has hardly been examined. The authors therefore decided to study this genotoxicity, including the frequency of sister chromatid exchange (SCE) of nine organic and three inorganic arsenic compounds. Observation of the metaphases in the SCE test revealed that only DMA of the organo- and inorgano-arsenic compounds induces tetraploids and mitotic arrest. This indicates that the role of DMA may be important in arsenic genotoxicity and may give a clue to the carcinogenic mechanism of arsenic.

  17. The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro.

    PubMed

    Carlsson, Johan A; Wold, Agnes E; Sandberg, Ann-Sofie; Östman, Sofia M

    2015-01-01

    Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c+ CD11b+ and CD11c+ CD11bneg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IAd remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violet low) and expressed CD69 or CD25, while more were necrotic (7AAD+). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4+ FoxP3+ CTLA-4+, CD4+ FoxP3+ Helios+ or CD4+ FoxP3+ PD-1+, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E2 while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells.

  18. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  19. Valproic acid induces the glutamate transporter excitatory amino acid transporter-3 in human oligodendroglioma cells.

    PubMed

    Bianchi, M G; Franchi-Gazzola, R; Reia, L; Allegri, M; Uggeri, J; Chiu, M; Sala, R; Bussolati, O

    2012-12-27

    Glutamate transport in early, undifferentiated oligodendrocytic precursors has not been characterized thus far. Here we show that human oligodendroglioma Hs683 cells are not endowed with EAAT-dependent anionic amino acid transport. However, in these cells, but not in U373 human glioblastoma cells, valproic acid (VPA), an inhibitor of histone deacetylases, markedly induces SLC1A1 mRNA, which encodes for the glutamate transporter EAAT3. The effect is detectable after 8h and persists up to 120h of treatment. EAAT3 protein increase becomes detectable after 24h of treatment and reaches its maximum after 72-96h, when it is eightfold more abundant than control. The initial influx of d-aspartate increases in parallel, exhibiting the typical features of an EAAT3-mediated process. SLC1A1 mRNA induction is associated with the increased expression of PDGFRA mRNA (+150%), a marker of early oligodendrocyte precursor cells, while the expression of GFAP, CNP and TUBB3 remains unchanged. Short term experiments have indicated that the VPA effect is shared by trichostatin A, another inhibitor of histone deacetylases. On the contrary, EAAT3 induction is neither prevented by inhibitors of mitogen-activated protein kinases nor triggered by a prolonged incubation with lithium, thus excluding a role for the GSK3β/β-catenin pathway. Thus, the VPA-dependent induction of the glutamate transporter EAAT3 in human oligodendroglioma cells likely occurs through an epigenetic mechanism and may represent an early indicator of commitment to oligodendrocytic differentiation.

  20. Effects of sodium bicarbonate on butyric acid-induced epithelial cell damage in vitro.

    PubMed

    Takigawa, Satoko; Sugano, Naoyuki; Ochiai, Kuniyasu; Arai, Noriyuki; Ota, Noriko; Ito, Koichi

    2008-12-01

    Butyric acid is detected in periodontal pockets and is thought to be involved in the initiation and progression of periodontal disease. We examined the effects of sodium bicarbonate on the butyric acid-induced epithelial cell damage. The human gingival carcinoma cell line Ca9-22 was cultured in medium that contained butyric acid with or without sodium bicarbonate. The viability of cells treated with sodium bicarbonate was significantly higher than that of cells treated with butyric acid alone. The effects of butyric acid on ICAM-1 expression were significantly improved by sodium bicarbonate. Within the limitations of this in vitro study, sodium bicarbonate was indicated to be a useful therapeutic agent to reduce the butyric acid-induced periodontal tissue damage.

  1. Ursolic acid suppresses leptin-induced cell proliferation in rat vascular smooth muscle cells.

    PubMed

    Yu, Ya-Mei; Tsai, Chiang-Chin; Tzeng, Yu-Wen; Chang, Weng-Cheng; Chiang, Su-Yin; Lee, Ming-Fen

    2017-01-29

    Accumulating lines of evidence indicate that high leptin levels are associated with adverse cardiovascular health in obese individuals. Proatherogenic effects of leptin include endothelial cell activation, vascular smooth muscle cell proliferation and migration. Ursolic acid (UA) has been reported to exhibit multiple biological effects including antioxidant and anti-inflammatory properties. In this study, we investigated the effect of UA on leptin-induced biological responses in rat vascular smooth muscle cells (VSMCs). A-10 VSMCs were treated with leptin in the presence or absence of UA. Intracellular reactive oxygen species (ROS) was probed by 2',7'-dichlorofluorescein diacetate. The expression of extracellular signal-regulated kinase (ERK)1/2, phospho-(ERK)1/2, nuclear factor-kappa B (NF-κB) p65 and p50, and matrix metalloproteinase-2 (MMP2) was determined by Western blotting. Immunocytochemistry and confocal laser scanning microscopy were also used for the detection of NF-κB. The secretion of MMP2 was detected by gelatin zymography. UA exhibited antioxidant activities in vitro. In rat VSMCs, UA effectively inhibited cell growth and the activity of MMP2 induced by leptin. These suppressive effects appeared by decreasing the activation of (ERK)1/2, the nuclear expression and translocation of NF-κB, and the production of ROS. UA appeared to inhibit leptin-induced atherosclerosis, which may prevent the development of obesity-induced cardiovascular diseases.

  2. Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells.

    PubMed

    Huang, Shang-Ming; Chuang, Hong-Chih; Wu, Chi-Hao; Yen, Gow-Chin

    2008-08-01

    In the process of glycation, methylglyoxal is a reactive dicarbonyl compound physiologically generated as an intermediate of glycolysis, and is found in high levels in blood or tissue of diabetic models. Biological glycation has been commonly implicated in the development of diabetic microvascular complications of neuropathy. Increasing evidence suggests that neuronal cell cycle regulatory failure followed by apoptosis is an important mechanism in the development of diabetic neuropathy complication. Naturally occurring antioxidants, especially phenolic acids have been recommended as the major bioactive compounds to prevent chronic diseases and promote health benefits. The objective of this study was to investigate the inhibitory abilities of phenolic acids (chlorogenic acid, syringic acid and vanillic acid) on methylglyoxal-induced mouse Neuro-2A neuroblastoma (Neuro-2A) cell apoptosis in the progression of diabetic neuropathy. The data indicated that methylglyoxal induced mouse Neuro-2A neuroblastoma (Neuro-2A) cell apoptosis via alternation of mitochondria membrane potential and Bax/Bcl-2 ratio, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase. Furthermore, the results demonstrated that activation of mitogen-activated protein kinase signal pathways (JNK and p38) participated in the methylglyoxal-induced Neuro-2A cell apoptosis process. Treatment of Neuro-2A cells with phenolic acids markedly suppresses cell apoptosis induced by methylglyoxal, suggesting that phenolic acids possess cytoprotective ability in the prevention of diabetic neuropathy complication.

  3. Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells.

    PubMed

    Luo, Yi; Rana, Payal; Will, Yvonne

    2012-06-01

    Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients.

  4. Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

    PubMed

    Raverdeau, Mathilde; Gely-Pernot, Aurore; Féret, Betty; Dennefeld, Christine; Benoit, Gérard; Davidson, Irwin; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

    2012-10-09

    Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

  5. Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid.

    PubMed

    An, Jung-Ung; Oh, Deok-Kun

    2013-09-01

    Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l(-1) yeast extract, 10 g l(-1) peptone, 5 g l(-1) oleic acid, 1 g l(-1) glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l(-1) hydroxy fatty acid, and 20 g l(-1) cells. Non-induced cells produced 38 g l(-1) γ-dodecalactone from 60 g l(-1) 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l(-1) h(-1) under the optimized conditions, whereas induced cells produced 51 g l(-1) γ-dodecalactone from 60 g l(-1) 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l(-1) h(-1). The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l(-1) γ-decalactone and 12 g l(-1) γ-butyrolactone from 60 g l(-1) 12-hydroxystearic acid and 60 g l(-1) 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.

  6. Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells

    SciTech Connect

    Luo, Yi Rana, Payal; Will, Yvonne

    2012-06-01

    Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients. -- Highlights: ► Palmitic acid and cyclosporine (CsA) synergistically increased cytotoxicity. ► The impairment of mitochondrial functions may contribute to the enhanced toxicity. ► Inhibition of JNK activity attenuated

  7. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    PubMed Central

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  8. Acid-inducible proton influx currents in the plasma membrane of murine osteoclast-like cells.

    PubMed

    Kuno, Miyuki; Li, Guangshuai; Moriura, Yoshie; Hino, Yoshiko; Kawawaki, Junko; Sakai, Hiromu

    2016-05-01

    Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na(+), Cl(-), and HCO3 (-), suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide. Extracellular Ca(2+) (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H(+) influx currents, which caused overshoot of the voltage-gated H(+) channels after removal of acids. The H(+) influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H(+) influx (H(+) leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.

  9. Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

    PubMed

    Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

    2014-01-01

    Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

  10. NDRG1 contributes to retinoic acid-induced differentiation of leukemic cells.

    PubMed

    Chen, Su; Han, Yu-Hui; Zheng, Ying; Zhao, Meng; Yan, Hua; Zhao, Qiao; Chen, Guo-Qiang; Li, Dao

    2009-08-01

    N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBPbeta) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation.

  11. Sasa quelpaertensis and p-coumaric acid attenuate oleic acid-induced lipid accumulation in HepG2 cells.

    PubMed

    Kim, Jeong-Hwan; Kang, Seong-Il; Shin, Hye-Sun; Yoon, Seon-A; Kang, Seung-Woo; Ko, Hee-Chul; Kim, Se-Jae

    2013-01-01

    In this study, we examined the effects of Jeju dwarf bamboo (Sasa quelpaertensis Nakai) extract (JBE) and p-coumaric acid (CA) on oleic acid (OA)-induced lipid accumulation in HepG2 cells. JBE and CA increased the phosphorylation of AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) and the expression of carnitine palmitoyl transferase 1a (CPT1a) in OA-treated HepG2 cells. Additionally, these compounds decreased sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and OA-induced lipid accumulation, suggesting that JBE and CA modulate lipid metabolism in HepG2 cells via the AMPK activation pathway.

  12. Uncoupling protein 2 regulates palmitic acid-induced hepatoma cell autophagy.

    PubMed

    Lou, Jiaxin; Wang, Yunjiao; Wang, Xuejiang; Jiang, Ying

    2014-01-01

    Mitochondrial uncoupling protein 2 (UCP2) is suggested to have a role in the development of nonalcoholic steatohepatitis (NASH). However, the mechanism remains unclear. Autophagy is an important mediator of many pathological responses. This study aims to investigate the relationship between UCP2 and hepatoma cells autophagy in palmitic acid- (PA-) induced lipotoxicity. H4IIE cells were treated with palmitic acid (PA), and cell autophagy and apoptosis were examined. UCP2 expression, in association with LC3-II and caspase-3, which are indicators of cell autophagy and apoptosis, respectively,was measured. Results demonstrated that UCP2 was associated with autophagy during PA-induced hepatic carcinoma cells injury. Tests on reactive oxygen species (ROS) showed that UCP2 overexpression strongly decreases PA-induced ROS production and apoptosis. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing enhances PA-induced ROS production and apoptosis. Autophagy partially participates in this progress. Moreover, UCP2 was associated with ATP synthesis during PA-induced autophagy. In conclusion, increasing UCP2 expression in hepatoma cells may contribute to cell autophagy and antiapoptotic as result of fatty acid injury. Our results may bring new insights for potential NASH therapies.

  13. Gallic acid induces HeLa cell death via increasing GSH depletion rather than ROS levels.

    PubMed

    Park, Woo Hyun

    2017-02-01

    Gallic acid (GA; 3,4,5-triphydroxyl-benzoic acid) is widely dispersed in various plants, fruits and foods and it shows various biological properties including anticancer effects. This study investigated the effects of GA on HeLa cervical cancer cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). GA dose-dependently inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC) at 24 or 72 h. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA induced apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). GA increased ROS levels including O2•- in HeLa cells at 24 h and it also induced GSH depletion. N-acetyl cysteine (NAC) increased the growth inhibition of GA-treated HeLa cells and enhanced the death of these cells. NAC differently influenced ROS levels in GA-treated HeLa cells and significantly increased GSH depletion in these cells. L-buthionine sulfoximine (BSO) increased MMP (∆ψm) loss, ROS levels and GSH depletion in GA-treated HeLa cells. In conclusion, GA significantly inhibited the growth of HeLa cells. GA-induced HeLa cell death was tightly related to GSH depletion rather than ROS level changes.

  14. A role for oxalic acid generation in ozone-induced signallization in Arabidopis cells.

    PubMed

    Tran, Daniel; Kadono, Takashi; Molas, Maria Lia; Errakhi, Rafik; Briand, Joël; Biligui, Bernadette; Kawano, Tomonori; Bouteau, François

    2013-03-01

    Ozone (O(3) ) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O(3) results in ascorbate-derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca(2+) concentration, generation of reactive oxygen species and cell death. We confirmed that O(3) reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O(3) -induced signalling pathways that trigger programmed cell death.

  15. The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity.

    PubMed

    Lolicato, Francesca; Brouwers, Jos F; de Lest, Chris H A van; Wubbolts, Richard; Aardema, Hilde; Priore, Paola; Roelen, Bernard A J; Helms, J Bernd; Gadella, Bart M

    2015-01-01

    Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.

  16. Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii.

    PubMed

    Ludovico, Paula; Sansonetty, Filipe; Silva, Manuel T; Côrte-Real, Manuela

    2003-03-01

    Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.

  17. Alpha-amylase production is induced by sulfuric acid in rice aleurone cells.

    PubMed

    Mitsunaga, Shin-ichiro; Kobayashi, Midori; Fukui, Satoe; Fukuoka, Kayoko; Kawakami, Osamu; Yamaguchi, Junji; Ohshima, Masahiro; Mitsui, Toshiaki

    2007-12-01

    The hydrolytic enzyme alpha-amylase (EC 3.2.1.1) is produced mainly in aleurone cells of germinating cereals, and the phytohormone gibberellin (GA) is essential for its induction. However, in rice (Oryza sativa L.), sulfuric acid (H(2)SO(4)) induces alpha-amylase production in aleurone tissue even in the absence of GA. Here, the pre-treatment of rice aleurone cells with H(2)SO(4) and incubation in water induced alpha-amylase activity, as if the cells had been incubated in GA solution.

  18. Protective effect of oleanolic acid on oxidized-low density lipoprotein induced endothelial cell apoptosis.

    PubMed

    Cao, Jianhua; Li, Guanghui; Wang, Meizhi; Li, Hui; Han, Zhiwu

    2015-10-01

    Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA) is a naturally-occurring triterpenoid with various promising pharmacological properties. The present study was conducted to determine the protective effects of OA against oxidized low-density lipoprotein (ox-LDL) induced endothelial cell apoptosis and the possible underlying mechanisms. Our results showed that ox-LDL significantly decreased cell viability and induced apoptosis in human umbilical vein endothelial cells (HUVECs). OA in the co-treatment showed a protective effect against ox-LDL induced loss in cell viability and an increase in apoptosis, which was associated with the modulating effect of OA on ox-LDL induced hypoxia-inducible factor 1α(HIF-1α) expression. Moreover, our results showed that the modulating effect of OA against ox-LDL induced HIF-1α expression was obtained via inhibition of lipoprotein receptor 1 (LOX-1)/reactive oxygen species (ROS) signaling. Collectively, we suggested that the protective effect of OA against ox-LDL induced HUVEC apoptosis might, at least in part, be obtained via inhibition of the LOX-1/ROS/HIF-1α signaling pathway.

  19. Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

    PubMed

    Jiao, Jiao; Sun, Ling; Zhou, Benguo; Gao, Zhengliang; Hao, Yu; Zhu, Xiaoping; Liang, Yuancun

    2014-08-15

    Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.

  20. Rosmarinic acid potentiates ATRA-induced macrophage differentiation in acute promyelocytic leukemia NB4 cells.

    PubMed

    Heo, Sook-Kyoung; Noh, Eui-Kyu; Yoon, Dong-Joon; Jo, Jae-Cheol; Koh, SuJin; Baek, Jin Ho; Park, Jae-Hoo; Min, Young Joo; Kim, Hawk

    2015-01-15

    Rosmarinic acid (RA, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid) has a number of biological activities, but little is known about anti-leukemic activities of RA combined with all-trans retinoic acid (ATRA) against acute promyelocytic leukemia (APL) cells. We examined the differentiation marker, CD11b, in bone marrow cells (BMC) of an APL patient, in NB4 cells (APL cell line), and in normal BMC and peripheral blood mononuclear cells (PBMC) of healthy subjects by flow cytometric analysis. ATRA/RA induced expression of CD11b in the BMC of the APL patient and in NB4 cells, but not in normal BMC or PBMC. Therefore, we realized that RA potentiated ATRA-induced macrophage differentiation in APL cells. Further characterization of the induced macrophages showed that they exhibited morphological changes and were able to phagocytose and generate reactive oxygen species. Th also had typical expression of C-C chemokine receptor type 1 (CCR1), CCR2, and intercellular adhesion molecule-1 (ICAM-1). Moreover, the expression of CD11b(+) and CD14(+) cells depended on ERK-NF-κB axis activation. Together, these results indicate that RA potentiates ATRA-induced macrophage differentiation in APL cells. Thus, RA may play an important role as an appurtenant differentiation agent for functional macrophage differentiation in APL. Additionally, the differentiated macrophages might have a normal life span and, they could die. These data indicate that co-treatment with RA and ATRA has potential as an anti-leukemic therapy in APL.

  1. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  2. Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

    PubMed Central

    Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

    2016-01-01

    Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

  3. Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells

    PubMed Central

    Gafar, Ahmed A.; Draz, Hossam M.; Goldberg, Alexander A.; Bashandy, Mohamed A.; Bakry, Sayed; Khalifa, Mahmoud A.; AbuShair, Walid; Titorenko, Vladimir I.

    2016-01-01

    Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI–LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively

  4. Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells.

    PubMed

    Gafar, Ahmed A; Draz, Hossam M; Goldberg, Alexander A; Bashandy, Mohamed A; Bakry, Sayed; Khalifa, Mahmoud A; AbuShair, Walid; Titorenko, Vladimir I; Sanderson, J Thomas

    2016-01-01

    Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI-LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively

  5. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

    PubMed Central

    Hopkins, Mandi M.; Zhang, Zhihong; Liu, Ze; Meier, Kathryn E.

    2016-01-01

    Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor. PMID:26821052

  6. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells.

    PubMed

    Hopkins, Mandi M; Zhang, Zhihong; Liu, Ze; Meier, Kathryn E

    2016-01-26

    Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor.

  7. Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

    SciTech Connect

    Qi Xinming; Cai Yan; Gong Likun; Liu Linlin; Chen Fangping; Xiao Ying; Wu Xiongfei; Li Yan; Xue Xiang |; Ren Jin . E-mail: cdser_simm@mail.shcnc.ac.cn

    2007-07-01

    Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca{sup 2+}, AAI caused mitochondrial swelling, leakage of Ca{sup 2+}, membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid.

  8. Ethacrynic acid and 1 alpha,25-dihydroxyvitamin D3 cooperatively inhibit proliferation and induce differentiation of human myeloid leukemia cells.

    PubMed

    Makishima, M; Honma, Y

    1996-09-01

    The active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 (VD3), inhibits proliferation and induces differentiation of leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. In this study we found that the loop diuretic ethacrynic acid, which is used to treat hypercalcemia, enhanced the differentiation of human leukemia cells induced by VD3. Ethacrynic acid alone inhibited the proliferation of human promyelocytic HL-60 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Ethacrynic acid effectively enhanced the growth-inhibiting action of VD3. In the presence of ethacrynic acid, VD3 increased the NBT-reducing and lysozyme activities and the CD11b expression of HL-60 cells more effectively than VD3 alone. Other loop diuretics, furosemide and bumetanide, also enhanced the differentiation of HL-60 cells induced by VD3, but to a lesser extent than ethacrynic acid. The differentiation of HL-60 cells induced by all-trans retinoic acid, dimethyl sulfoxide or phorbol-12-myristate 13-acetate was also enhanced by ethacrynic acid with increasing NBT-reducing and lysozyme activities and the expression of CD11b or CD14 surface antigen. Morphologically, ethacrynic acid enhanced the monocytic differentiation of HL-60 cells induced by VD3 and phorbol ester and the granulocytic differentiation by retinoic acid and dimethyl sulfoxide. Other human myelomonocytic leukemia ML-1, U937, P39/TSU and P31/FUJ cells were induced to differentiate by VD3 and this was also enhanced by ethacrynic acid. The long-term culture of HL-60 cells showed that ethacrynic acid plus VD3 induced the complete growth arrest of HL-60 cells. Therefore ethacrynic acid, which is used to treat hypercalcemia, enhanced the proliferation-inhibiting and differentiation-inducing activities of VD3 and the combination of ethacrynic acid and VD3 may be useful in therapy for myeloid leukemia.

  9. Potassium loss is involved in tobacco cell death induced by palmitoleic acid and ceramide.

    PubMed

    Peters, Jeanne; Chin, Chee-Kok

    2007-09-01

    Tobacco cell death induced by palmitoleic acid (16:1), ceramide, and KCN was found to possess features associated with program cell death (PCD), including cell volume decrease, loss of membrane integrity, DNA damage, nuclear and plastid disorganization, and chromatin condensation. Cell volume decrease was found to be caused by loss of intracellular K(+). Ba(2+) was able to prevent the K(+) loss and it also protected the cells from death induced by 16:1 and ceramide but not KCN. The results suggest that K(+) loss is a critical step in plant PCD. The inability of Ba(2+) to prevent cell death was most likely due to its other effects of KCN, i.e., inhibition of cytochrome oxidase in the respiratory chain and generation of reactive oxygen species.

  10. Polyunsaturated fatty acid enrichment enhances endothelial cell-induced low-density-lipoprotein peroxidation.

    PubMed Central

    Mazière, C; Dantin, F; Conte, M A; Degonville, J; Ali, D; Dubois, F; Mazière, J C

    1998-01-01

    Oxidative modification of low-density lipoprotein (LDL) is an important feature in the initiation and progression of atherosclerosis. LDL modification by endothelial cells was studied after supplementation of the cells with oleic acid and polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series. In terms of the lipid peroxidation product [thiobarbituric acid reactive substances (TBARS)] content and diene level of the LDL particle, oleic acid had no significant effect, and linoleic acid was poorly effective. Gamma linolenic acid (C18:3,n-6) and arachidonic acid (C20:4,n-6) increased by about 1.6-1.9-fold the cell-mediated LDL modification. PUFA from the n-3 series, alpha linolenic acid (C18:3,n-3), eicosapentaenoic acid (C20:5,n-3) and docosahexaenoic acid (C22:6,n-3), induced a less marked effect (1. 3-1.6-fold increase). The relative electrophoretic mobility of the LDL particle and its degradation by macrophages were enhanced in parallel. Concomitantly, PUFA stimulated superoxide anion secretion by endothelial cells. The intracellular TBARS content was also increased by PUFA. Comparison of PUFA from the two series indicates a good correlation between LDL oxidative modification, superoxide anion secretion and intracellular lipid peroxidation. The lipophilic antioxidant vitamin E decreased the basal as well as the PUFA-stimulated LDL peroxidation. These results indicate that PUFAs with a high degree of unsaturation of the n-6 and n-3 series could accelerate cell-mediated LDL peroxidation and thus aggravate the atherosclerotic process. PMID:9806884

  11. Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells.

    PubMed

    Ignacio Barrasa, Juan; Olmo, Nieves; Pérez-Ramos, Pablo; Santiago-Gómez, Angélica; Lecona, Emilio; Turnay, Javier; Antonia Lizarbe, M

    2011-10-01

    The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

  12. Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

    PubMed Central

    Fernández-Sánchez, Laura; Rondón, Netxibeth; Esquiva, Gema; Germain, Francisco; de la Villa, Pedro; Cuenca, Nicolás

    2015-01-01

    Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss. PMID:26379056

  13. Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration.

    PubMed

    Gómez-Vicente, Violeta; Lax, Pedro; Fernández-Sánchez, Laura; Rondón, Netxibeth; Esquiva, Gema; Germain, Francisco; de la Villa, Pedro; Cuenca, Nicolás

    2015-01-01

    Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.

  14. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    PubMed Central

    Dong, Xue-Jun; Zhang, Guo-Rong; Zhou, Qing-Jun; Pan, Ruo-Lang; Chen, Ye; Xiang, Li-Xin; Shao, Jian-Zhong

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  15. Phenylacetate synergizes with retinoic acid in inducing the differentiation of human neuroblastoma cells.

    PubMed

    Sidell, N; Wada, R; Han, G; Chang, B; Shack, S; Moore, T; Samid, D

    1995-02-08

    Phenylacetate, a natural metabolite of phenylalanine which was originally described as a plant growth hormone, has recently gained attention as a possible differentiation inducer for a variety of human tumor cell types. This interest prompted us to assess the ability of sodium phenylacetate (NaPA) to promote the differentiation of human neuroblastoma cells, both alone and in combination with retinoic acid (RA), a known inducer of neuroblastoma differentiation and maturation. Using the LA-N-5 cell line, we have determined that NaPA can stimulate the differentiation of neuroblastoma cells, as evidenced by dose-dependent inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity and reduction of N-myc expression. Furthermore, NaPA and RA synergized in inducing differentiation, in that combination treatment resulted in cessation of cell growth along with morphologic and biochemical changes indicative of the loss of malignant properties. We have determined that NaPA can markedly enhance mRNA levels of the nuclear RA receptor-beta (RAR beta) in LA-N-5 cells prior to morphologic or other phenotypic changes induced by this compound. This effect appeared to be distinct from the ability of NaPA to alter tumor cell lipid metabolism via inhibition of protein isoprenylation. Thus among its varied effects on LA-N-5 cells, NaPA appears to interact with the RA pathway at the nuclear level by up-regulating RAR beta expression.

  16. CCN1 is critical for acid-induced esophageal epithelial cell transformation.

    PubMed

    Modak, Cristina; Mouazzen, Wasim; Narvaez, Reinier; Reavis, Kevin M; Chai, Jianyuan

    2010-02-19

    CCN1 is a matricellular protein involved in both wound healing and cancer cell invasion. Increased CCN1 expression has been associated with the development of Barrett's esophagus and the increased risk of progression to esophageal adenocarcinoma. In both cases, acid reflux is a major contributor. Low pH has been shown to induce CCN1 gene expression in esophageal epithelial cells. Here we demonstrated that both CCN1 and low pH could cause esophageal epithelial cell transformation, including loss of E-cadherin, disruption of cell-cell junctions, and expression of mesenchymal markers. Furthermore, knockdown of CCN1 through RNA interference sufficiently attenuated acid-driven cell phenotypic changes, while over-expression of CCN1 exacerbated these effects, indicating a critical role of CCN1 in acid-induced esophageal epithelial cell transformation. Given the pivotal role of low pH in gastro-esophageal reflux disease and its progression towards esophageal adenocarcinoma, our study identified CCN1 as a key molecule mediating this process.

  17. Corosolic acid inhibits the proliferation of osteosarcoma cells by inducing apoptosis

    PubMed Central

    Jia, Yong; Yuan, Hua; Shan, Shouqin; Xu, Gang; Yu, Jie; Zhao, Chenguang; Mou, Xiang

    2016-01-01

    Corosolic acid (CRA), a pentacyclic triterpene isolated from medicinal herbs, has been reported to exhibit anticancer properties in several cancers. However, the anticancer activity of CRA in osteosarcoma cells is still unclear. In the present study, the inhibitory effect of CRA in osteosarcoma MG-63 cells was investigated, and the results revealed that CRA significantly inhibited the viability of MG-63 cells in a dose- and time-dependent manner. A typical apoptotic hallmark such as DNA ladder was detected by agarose gel electrophoresis following treatment with CRA. Further experiments demonstrated that CRA induced apoptosis of MG-63 cells by flow cytometry using propidium iodide and annexin V staining. In addition, it was observed that the apoptosis of MG-63 cells induced by CRA was closely associated with activation of caspase-3 and caspase-9, loss of mitochondrial membrane potential, and release of cytochrome c from mitochondria, suggesting that CRA may trigger the activation of the mitochondria-mediated apoptosis pathway. In addition, the inhibition of caspase activity attenuated the CRA-induced apoptosis of MG-63 cells, which further confirmed the role of the mitochondrial pathway in CRA-induced apoptosis. These results indicated that CRA could induce the apoptosis of osteosarcoma cells through activating the mitochondrial pathway, which provides an evidence that CRA may be a useful chemotherapeutic agent for osteosarcoma. PMID:27895790

  18. Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells.

    PubMed

    Jin, Un-Ho; Song, Kwon-Ho; Motomura, Muneo; Suzuki, Ikukatsu; Gu, Yeun-Hwa; Kang, Yun-Jeong; Moon, Tae-Chul; Kim, Cheorl-Ho

    2008-03-01

    Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

  19. Lack of kainic acid-induced gamma oscillations predicts subsequent CA1 excitotoxic cell death

    PubMed Central

    Jinde, Seiichiro; Belforte, Juan E.; Yamamoto, Jun; Wilson, Matthew A.; Tonegawa, Susumu; Nakazawa, Kazu

    2009-01-01

    Gamma oscillations are a prominent feature of hippocampal network activity, but their functional role remains debated, ranging from mere epiphenomenon to crucial for information processing. Similarly, persistent gamma oscillations sometimes appear prior to epileptic discharges in patients with mesial temporal sclerosis. However, the significance of this activity in hippocampal excitotoxicity is unclear. We assessed the relationship between kainic acid (KA)-induced gamma oscillations and excitotoxicity in genetically-engineered mice in which N-methyl-D-aspartic acid (NMDA) receptor deletion was confined to CA3 pyramidal cells. Mutants showed reduced CA3 pyramidal cell firing and augmented sharp wave-ripple activity, resulting in higher susceptibility to KA-induced seizures, and leading to strikingly selective neurodegeneration in the CA1 subfield. Interestingly, the KA-induced gamma-aminobutyric acid (GABA) level increases and persistent 30-50 Hz gamma oscillations observed in control mice prior to the first seizure discharge was abolished in the mutants. Consequently, on subsequent days, mutants manifested prolonged epileptiform activity and massive neurodegeneration of CA1 cells, including local GABAergic neurons. Remarkably, pretreatment with the potassium channel blocker α-dendrotoxin (DTX) increased GABA levels, restored gamma oscillations, and prevented CA1 degeneration in the mutants. These results demonstrate that emergence of low frequency gamma oscillations predicts increased resistance to KA-induced excitotoxicity, raising the possibility that gamma oscillations may have potential prognostic value for the treatment of epilepsy. PMID:19735292

  20. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    PubMed

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  1. Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures.

    PubMed

    Kapulnik, Y; Yalpani, N; Raskin, I

    1992-12-01

    Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O(2) uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O(2) uptake by up to 40% as compared to controls. Application of 20 mum salicylic acid (SA) to young cells increased cyanide-resistant O(2) uptake within 2 h. Development of KCN resistance did not affect total O(2) uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O(2) consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.

  2. Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

    PubMed Central

    Thiele, C J; Cohen, P S; Israel, M A

    1988-01-01

    We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription. Images PMID:3380093

  3. Betulin, betulinic acid and butein are inhibitors of acetaldehyde-induced activation of liver stellate cells.

    PubMed

    Szuster-Ciesielska, Agnieszka; Plewka, Krzysztof; Kandefer-Szerszeń, Martyna

    2011-01-01

    Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids; however, the activity of other triterpenes like betulin and betulinic acid has not been examined. Butein has also been reported to prevent and partly reverse liver fibrosis in vivo, although its mechanism of action is poorly understood. Therefore, the aim of this study was to determine the antifibrotic potential of butein, betulin, and betulinic acid and examine their mechanisms of action in vitro. This study was conducted in rat stellate cells (HSCs) that were treated with acetaldehyde, which is the most reactive product of ethanol metabolism. Butein, betulin, and betulinic acid were preincubated with rat HSCs at non-toxic concentrations. Treatment effects were measured in regard to acetaldehyde-induced toxicity and cell migration, and several markers of HSC activation were evaluated, including smooth muscle α-actin (α-SMA) and procollagen I expression. In addition, changes in the release of reactive oxygen species (ROS) and cytokines such as tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1) and changes in the production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were determined. In vitro, HSCs were protected against acetaldehyde-induced toxicity by betulin but not by betulinic acid and butein. However, butein, betulin, and betulinic acid inhibited the production of ROS by HSCs treated with acetaldehyde and inhibited their migration. Butein also inhibited acetaldehyde-induced TGF-β1 production. Butein, betulin, and betulinic acid down-regulated acetaldehyde-induced production of TIMP-1 and TIMP-2. Betulin decreased the acetaldehyde-induced activity of MMP-2, but butein and betulinic acid did not. The results indicated that butein, betulin, and betulinic acid inhibited the acetaldehyde-induced activation of HSCs. Each drug functioned in a different manner, whereby some were acting as either

  4. Ethylene signaling in salt stress- and salicylic acid-induced programmed cell death in tomato suspension cells.

    PubMed

    Poór, Péter; Kovács, Judit; Szopkó, Dóra; Tari, Irma

    2013-02-01

    Salt stress- and salicylic acid (SA)-induced cell death can be activated by various signaling pathways including ethylene (ET) signaling in intact tomato plants. In tomato suspension cultures, a treatment with 250 mM NaCl increased the production of reactive oxygen species (ROS), nitric oxide (NO), and ET. The 10(-3) M SA-induced cell death was also accompanied by ROS and NO production, but ET emanation, the most characteristic difference between the two cell death programs, did not change. ET synthesis was enhanced by addition of ET precursor 1-aminocyclopropane-1-carboxylic acid, which, after 2 h, increased the ROS production in the case of both stressors and accelerated cell death under salt stress. However, it did not change the viability and NO levels in SA-treated samples. The effect of ET induced by salt stress could be blocked with silver thiosulfate (STS), an inhibitor of ET action. STS reduced the death of cells which is in accordance with the decrease in ROS production of cells exposed to high salinity. Unexpectedly, application of STS together with SA resulted in increasing ROS and reduced NO accumulation which led to a faster cell death. NaCl- and SA-induced cell death was blocked by Ca(2+) chelator EGTA and calmodulin inhibitor W-7, or with the inhibitors of ROS. The inhibitor of MAPKs, PD98059, and the cysteine protease inhibitor E-64 reduced cell death in both cases. These results show that NaCl induces cell death mainly by ET-induced ROS production, but ROS generated by SA was not controlled by ET in tomato cell suspension.

  5. Ascorbic acid protects against colistin sulfate-induced neurotoxicity in PC12 cells.

    PubMed

    Liu, Yang; Dai, Chongshan; Gao, Ruixia; Li, Jichang

    2013-10-01

    This study aimed to examine the protective effect of ascorbic acid against colistin-induced neurotoxicity mediated by oxidative stress, a potential mechanism. An in vitro neurotoxicity model was established with PC12 cells exposed to 125 µg/mL colistin sulfate for 24 h. PC12 cells were treated with colistin (125 µg/mL) in the absence and presence of ascorbic acid (0.1, 1.0 and 10 µM/mL) for 24 h. Both 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay were carried out to evaluate cell viability. The levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) levels were assessed. Moreover, we tested the level of DNA fragmentation, the release of cytochrome-c and the expressions of caspase-9 and -3 mRNA. The results showed that 1 and 10 µM/mL ascorbic acid significantly increased the cell viability and the levels of SOD and GSH (both p<0.05), while 0.1, 1 and 10 µM/mL ascorbic acid significantly decreased the generation of ROS, the release of cytochrome-c, formation of DNA fragmentation and the expressions of caspase-9 and -3 mRNA in colistin-treated PC12 cells, compared with the colistin model group. These results suggest that ascorbic acid could reduce colistin sulfate-induced neurotoxicity through the resistance of oxidative stress and the prevention of apoptosis mediated via mitochondria pathway. They also highlight the potential of coadministering ascorbic acid to widen the therapeutic dose of colistin.

  6. Protective Effect of Edaravone in Primary Cerebellar Granule Neurons against Iodoacetic Acid-Induced Cell Injury

    PubMed Central

    Zhou, Xinhua; Zhu, Longjun; Wang, Liang; Guo, Baojian; Zhang, Gaoxiao; Sun, Yewei; Zhang, Zaijun; Lee, Simon Ming-Yuen; Yu, Pei; Wang, Yuqiang

    2015-01-01

    Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal death in vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities. PMID:26557222

  7. Ursodeoxycholic acid protects colon cancer HCT116 cells from deoxycholic acid-induced apoptosis by inhibiting apoptosome formation.

    PubMed

    Saeki, Tohru; Yui, Satoko; Hirai, Tadashi; Fujii, Takami; Okada, Sawami; Kanamoto, Ryuhei

    2012-01-01

    We previously demonstrated that ursodeoxycholic acid (UDC) requires prolonged (≥5 h) preincubation to exhibit effective protection of colon cancer HCT116 cells from deoxycholic acid (DC)-induced apoptosis. Although UDC diminished DC-mediated caspase-9 activation, cytochrome c release from the mitochondria was not inhibited, indicating that UDC acts on the steps of caspase-9 activation. In the present study, therefore, we investigated the effects of UDC on the factors involved in caspase-9 activation. We found that UDC had no significant effect on the expression of antiapoptotic XIAP. Furthermore, UDC did not affect the expression or release of proapoptotic Smac/DIABLO, or the association of XIAP and Smac/DIABLO. In contrast, association of Apaf-1 and caspase-9 stimulated by 500 μM DC was inhibited by UDC pretreatment. Although UDC caused remarkable activation of Akt/PKB, phosphatidylinositol-3-kinase (PI3K) inhibitor did not significantly reduce UDC-mediated cytoprotection. Furthermore, phosphorylation of threonine residues on caspase-9 after UDC pretreatment could not be detected. UDC-mediated cytoprotection was independent of the MAPK pathway, and cyclic AMP (cAMP) analogue did not inhibit DC-induced apoptosis. Our results indicate that UDC protects colon cancer cells from apoptosis induced by hydrophobic bile acids, by inhibiting apoptosome formation independently of the survival signals mediated by the PI3K, MAPK, or cAMP pathways.

  8. Inhibition of monomethylarsonous acid (MMA(III))-induced cell malignant transformation through restoring dysregulated histone acetylation.

    PubMed

    Ge, Yichen; Gong, Zhihong; Olson, James R; Xu, Peilin; Buck, Michael J; Ren, Xuefeng

    2013-10-04

    Inorganic arsenic (iAs) and its high toxic metabolite, monomethylarsonous acid (MMA(III)), are able to induce malignant transformation of human cells. Chronic exposure to these chemicals is associated with an increased risk of developing multiple cancers in human. However, the mechanisms contributing to iAs/MMA(III)-induced cell malignant transformation and carcinogenesis are not fully elucidated. We recently showed that iAs/MMA(III) exposure to human cells led to a decreased level of histone acetylation globally, which was associated with an increased sensitivity to arsenic cytotoxicity. In the current study, it demonstrated that prolonged exposure to low-level MMA(III) in human urothelial cells significantly increased the expression and activity of histone deacetylases (HDACs) with an associated reduction of histone acetylation levels both globally and lysine specifically. Administration of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), at 4 weeks after the initial MMA(III) treatment inhibited the MMA(III)-mediated up-regulation of the expression and activities of HDACs, leading to increase histone acetylation and prevention of MMA(III)-induced malignant transformation. These new findings suggest that histone acetylation dysregulation may be a key mechanism in MMA(III)-induced malignant transformation and carcinogenesis, and that HDAC inhibitors could be targeted to prevent or treat iAs-related cancers.

  9. Inhibition of TNF-alpha induced cell death in human umbilical vein endothelial cells and Jurkat cells by protocatechuic acid.

    PubMed

    Zhou-Stache, J; Buettner, R; Artmann, G; Mittermayer, C; Bosserhoff, A K

    2002-11-01

    The Chinese herb radix Salviae miltiorrhizae (RSM) is used in traditional Chinese medicine as a treatment for cardiovascular and cerebrovascular diseases. Several components of the plant extract from salvia mitorrhiza bunge have been determined previously, one of which is protocatechuic acid (PAC). It has been found, in the study, that PAC inhibited TNF-alpha-induced cell death of human umbilical vein endothelial cells (HUVECs) and Jurkat cells in a concentration of 100 microM when applied 2 h prior to TNF-alpha exposure. Molecular studies revealed that PAC activated NF-kappaB with a maximum effect after 30 min of treatment. Inhibition of NF-kappaB action by MG132 and NF-kappaB inhibitory peptide suppressed the cell-protective effect of PAC. Further, degradation of IkBalpha occurred in response to PAC treatment. The results provide evidence that activation of NF-kappaB plays an important role in mediating the cell-protecting effect of PAC on HUVECs and Jurkat cells. Further studies are required to test whether PAC, a component of radix salviae miltiorrhizae, could be useful in preventing in vivo cell death resulting from cardiovascular or cerebrovascular diseases.

  10. Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells.

    PubMed

    Romanov, Victor; Whyard, Terry; Bonala, Radha; Johnson, Francis; Grollman, Arthur

    2011-12-01

    Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5'-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.

  11. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    PubMed

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-01-25

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  12. Mast cell mediators in citric acid-induced airway constriction of guinea pigs

    SciTech Connect

    Lin, C.-H.; Lai, Y.-L. . E-mail: tiger@ha.mc.ntu.edu.tw

    2005-08-15

    We demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. In this study, we further investigated the underlying mediator(s) for this type of airway constriction. At first, to examine effects caused by blocking agents, 67 young Hartley guinea pigs were divided into 7 groups: saline + CA; methysergide (serotonin receptor antagonist) + CA; MK-886 (leukotriene synthesis inhibitor) + CA; mepyramine (histamine H{sub 1} receptor antagonist) + CA; indomethacin (cyclooxygenase inhibitor) + CA; cromolyn sodium (mast cell stabilizer) + CA; and compound 48/80 (mast cell degranulating agent) + CA. Then, we tested whether leukotriene C{sub 4} (LTC{sub 4}) or histamine enhances CA-induced airway constriction in compound 48/80-pretreated guinea pigs. We measured dynamic respiratory compliance (Crs) and forced expiratory volume in 0.1 s (FEV{sub 0.1}) during either baseline or recovery period. In addition, we detected histamine level, an index of pulmonary mast cell degranulation, in bronchoalveolar lavage (BAL) samples. Citric acid aerosol inhalation caused decreases in Crs and FEV{sub 0.1}, indicating airway constriction in the control group. This airway constriction was significantly attenuated by MK-886, mepyramine, cromolyn sodium, and compound 48/80, but not by either methysergide or indomethacin. Both LTC{sub 4} and histamine infusion significantly increased the magnitude of CA-induced airway constriction in compound 48/80-pretreated guinea pigs. Citric acid inhalation caused significant increase in histamine level in the BAL sample, which was significantly suppressed by compound 48/80. These results suggest that leukotrienes and histamine originating from mast cells play an important role in CA inhalation-induced noncholinergic airway constriction.

  13. Mast cells in citric acid-induced cough of guinea pigs

    SciTech Connect

    Lai, Y.-L. . E-mail: tiger@ha.mc.ntu.edu.tw; Lin, T.-Y.

    2005-01-01

    It was demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. To investigate the role of mast cells in CA-induced cough, three experiments were carried out in this study. In the first experiment, 59 guinea pigs were employed and we used compound 48/80 to deplete mast cells, cromolyn sodium to stabilize mast cells, MK-886 to inhibit leukotriene synthesis, pyrilamine to antagonize histamine H{sub 1} receptor, methysergide to antagonize serotonin receptor, and indomethacin to inhibit cyclooxygenase. In the second experiment, 56 compound 48/80-pretreated animals were divided into two parts; the first one was used to test the role of exogenous leukotriene (LT) C{sub 4}, while the second one to test the role of exogenous histamine in CA-induced cough. Each animal with one of the above pretreatments was exposed sequentially to saline (baseline) and CA (0.6 M) aerosol, each for 3 min. Then, cough was recorded for 12 min using a barometric body plethysmograph. In the third experiment, the activation of mast cells upon CA inhalation was investigated by determining arterial plasma histamine concentration in 17 animals. Exposure to CA induced a marked increase in cough number. Compound 48/80, cromolyn sodium, MK-886 and pyrilamine, but not indomethacin or methysergide, significantly attenuated CA-induced cough. Injection of LTC{sub 4} or histamine caused a significant increase in CA-induced cough in compound 48/80-pretreated animals. In addition, CA inhalation caused significant increase in plasma histamine concentration, which was blocked by compound 48/80 pretreatment. These results suggest that mast cells play an important role in CA aerosol inhalation-induced cough via perhaps mediators LTs and histamine.

  14. Beneficial effects of carnosic acid on dieldrin-induced dopaminergic neuronal cell death.

    PubMed

    Park, Jeong Ae; Kim, Seung; Lee, Sook-Young; Kim, Chun-Sung; Kim, Do Kyung; Kim, Sung-Jun; Chun, Hong Sung

    2008-08-27

    Carnosic acid (CA) is one of the bioactive polyphenols present in extracts of the herb rosemary (Rosmarinus officinalis). In this study, we examined possible protective effects of CA on neurotoxicity induced by dieldrin, an organochlorine pesticide implicated in sporadic Parkinson's disease, in cultured dopaminergic cells (SN4741). CA (5-10 muM) pretreatment showed potent protective effects in a concentration-related manner and prevented dieldrin (10 muM)-induced caspase-3 activation, Jun N-terminal kinase phosphorylation, and caspase-12 activation. Furthermore, dieldrin-induced downregulation of brain-derived neurotrophic factor production was significantly attenuated by CA. These results suggest that CA may safeguard dopaminergic neuronal cells from environmental neurotoxins by enhancing brain-derived neurotrophic factor and repressing apoptotic molecules.

  15. Koetjapic acid, a natural triterpenoid, induces apoptosis in colon cancer cells.

    PubMed

    Nassar, Zeyad D; Aisha, Abdalrahim F A; Idris, Norshirin; Khadeer Ahamed, Mohamed B; Ismail, Zhari; Abu-Salah, Khalid M; Alrokayan, Salman A; Shah Abdul Majid, Amin Malik

    2012-03-01

    Deregulated cell signaling pathways result in cancer development. More than one signal transduction pathway is involved in colorectal cancer pathogenesis and progression. Koetjapic acid (KA) is a naturally occurring seco-A-ring oleanene triterpene isolated from the Sandoricum koetjape stem bark. We report the cellular and molecular mechanisms of anticancer activity of KA towards human colorectal cancer. The results showed that KA induces apoptosis in HCT 116 colorectal carcinoma cells by inducing the activation of extrinsic and intrinsic caspases. We confirmed that KA-induced apoptosis was mediated by DNA fragmentation, nuclear condensation and disruption in the mitochondrial membrane potential. Further studies on the effect of KA on cancer pathways show that the compound causes down-regulation of Wnt, HIF-1α, MAP/ERK/JNK and Myc/Max signaling pathways and up-regulates the NF-κB signaling pathway. The result of this study highlights the anticancer potential of KA against colorectal cancer.

  16. Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury*

    PubMed Central

    Samad, Aysha; James, Andrew; Wong, James; Mankad, Parini; Whitehouse, John; Patel, Waseema; Alves-Simoes, Marta; Siriwardena, Ajith K.; Bruce, Jason I. E.

    2014-01-01

    Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis. PMID:24993827

  17. Lipoic acid induces p53-independent cell death in colorectal cancer cells and potentiates the cytotoxicity of 5-fluorouracil.

    PubMed

    Dörsam, Bastian; Göder, Anja; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-10-01

    Alpha-lipoic acid (LA), which plays a pivotal role in mitochondrial energy metabolism, is an endogenous dithiol compound with an array of antioxidative functions. It has been shown that LA triggers cell death in tumor cell lines, whereas non-transformed cells are hardly affected. In the present study, we analyzed the cytotoxicity of LA on colorectal cancer (CRC) cells differing in their p53 status and investigated a putative synergistic effect with the anticancer drug 5-fluorouracil (5-FU). We show that LA induces a dose-dependent decrease in cell viability, which was independent of the p53 status as attested in isogenic p53-proficient and p53-deficient cell lines. This effect was largely attributable to cell death induction as revealed by Annexin-V/PI staining. LA-treated HCT116 cells underwent caspase-dependent and caspase-independent cell death, which was blocked by the pan-caspase inhibitor zVAD and the RIP-kinase inhibitor Necrostatin-1, respectively. In CaCO-2 and HT29 cells, LA induced caspase-dependent cell demise via activation of caspase-9, caspase-3 and caspase-7 with subsequent PARP-1 cleavage as demonstrated by immunoblot analysis, activity assays and pan-caspase inhibition. Interestingly, LA treatment did neither activate p53 nor induced genotoxic effects as shown by lack of DNA strand breaks and phosphorylation of histone 2AX. Finally, we provide evidence that LA increases the cytotoxic effect induced by the anticancer drug 5-FU as revealed by significantly enhanced cell death rates in HCT116 and CaCO-2 cells. Collectively, these findings demonstrate that LA induces CRC cell death independent of their p53 status and potentiates the cytotoxicity of 5-FU without causing DNA damage on its own, which makes it a candidate for tumor therapy.

  18. [Apoptosis of retinoic acid resistant NB4-R1 cells induced with curcumin and its mechanism].

    PubMed

    Zhang, Zhang-Lin; Kong, Yun-Yuan; Wan, La-Gen

    2010-04-01

    This study was purposed to explore the inhibitory effect of Curcumin on growth of retinoic acid-resistant acute promyelocytic leukemia (APL) cells and its mechanism. The NB4-R1, an APL cell line resistant to retinoic acid, was used as a model. The growth level of NB4-R1 was detected by MTT assay, the morphologic features of cells were observed by light microscopy, the mitochondrial transmembrane potential was determined by flow cytometry, the expressions of apoptosis-related proteins procaspase 3, caspase 3, PARP and BCL-XL were measured by Western blot. The results indicated that the sensitivity of NB4-R1 to Curcumin was consistent with NB4 though NB4-R1 was resistant to retinoic acid, Curcumin displayed inhibitory effect on growth of NB4-R1 in time-and concentration-dependent manners. The morphologic observation showed existence of apoptotic bodies in NB4-R1 cells treated with 20 micromol/L of Curcumin. The flow cytometry indicated that the mitochondrial transmembrane potential in NB4-R1 cells treated with 20 micromol/L of Curcumin obviously decreased. The Western blot detection revealed that expressions of pro-caspase 3 and BCL-XL were down-regulated, expressions of caspase 3 and sheared PAPP were up-regulated in NB4-R1 cells treated with 20 micromol/L of Curcumin. It is concluded that the Curcumin can inhibit the growth and induce the apoptosis of NB4-R1.

  19. Interplay between cell cycle and autophagy induced by boswellic acid analog

    PubMed Central

    Pathania, Anup S.; Guru, Santosh K.; Kumar, Suresh; Kumar, Ashok; Ahmad, Masroor; Bhushan, Shashi; Sharma, Parduman R.; Mahajan, Priya; Shah, Bhahwal A.; Sharma, Simmi; Nargotra, Amit; Vishwakarma, Ram; Korkaya, Hasan; Malik, Fayaz

    2016-01-01

    In this study, we investigated the role of autophagy induced by boswellic acid analog BA145 on cell cycle progression in pancreatic cancer cells. BA145 induced robust autophagy in pancreatic cancer cell line PANC-1 and exhibited cell proliferation inhibition by inducing cells to undergo G2/M arrest. Inhibition of G2/M progression was associated with decreased expression of cyclin A, cyclin B, cyclin E, cdc2, cdc25c and CDK-1. Pre-treatment of cells with autophagy inhibitors or silencing the expression of key autophagy genes abrogated BA145 induced G2/M arrest and downregulation of cell cycle regulatory proteins. It was further observed that BA145 induced autophagy by targeting mTOR kinase (IC50 1 μM), leading to reduced expression of p-mTOR, p-p70S6K (T389), p-4EBP (T37/46) and p-S6 (S240/244). Notably, inhibition of mTOR signalling by BA145 was followed by attendant activation of AKT and its membrane translocation. Inhibition of Akt through pharmacological inhibitors or siRNAs enhanced BA145 mediated autophagy, G2/M arrest and reduced expression of G2/M regulators. Further studies revealed that BA145 arbitrated inhibition of mTOR led to the activation of Akt through IGFR/PI3k/Akt feedback loop. Intervention in IGFR/PI3k/Akt loop further depreciated Akt phosphorylation and its membrane translocation that culminates in augmented autophagy with concomitant G2/M arrest and cell death. PMID:27680387

  20. Gambogic acid inhibits growth, induces apoptosis, and overcomes drug resistance in human colorectal cancer cells

    PubMed Central

    WEN, CHUANGYU; HUANG, LANLAN; CHEN, JUNXIONG; LIN, MENGMENG; LI, WEN; LU, BIYAN; RUTNAM, ZINA JEYAPALAN; IWAMOTO, AIKICHI; WANG, ZHONGYANG; YANG, XIANGLING; LIU, HUANLIANG

    2015-01-01

    The emergence of chemoresistance is a major limitation of colorectal cancer (CRC) therapies and novel biologically based therapies are urgently needed. Natural products represent a novel potential anticancer therapy. Gambogic acid (GA), a small molecule derived from Garcinia hanburyi Hook. f., has been demonstrated to be highly cytotoxic to several types of cancer cells and have low toxicity to the hematopoietic system. However, the potential role of GA in colorectal cancer and its ability to overcome the chemotherapeutic resistance in CRC cells have not been well studied. In the present study, we showed that GA directly inhibited proliferation and induced apoptosis in both 5-fluorouracil (5-FU) sensitive and 5-FU resistant colorectal cancer cells; induced apoptosis via activating JNK signaling pathway. The data, therefore, suggested an alternative strategy to overcome 5-FU resistance in CRC and that GA could be a promising medicinal compound for colorectal cancer therapy. PMID:26397804

  1. Effect of caffeic acid esters on carcinogen-induced mutagenicity and human colon adenocarcinoma cell growth.

    PubMed

    Rao, C V; Desai, D; Kaul, B; Amin, S; Reddy, B S

    1992-11-16

    Propolis, a honey bee hive product, is thought to exhibit a broad spectrum of activities including antibiotic, antiviral, anti-inflammatory and tumor growth inhibition; some of the observed biological activities may be due to caffeic acid (cinnamic acid) esters that are present in propolis. In the present study we synthesized three caffeic acid esters, namely methyl caffeate (MC), phenylethyl caffeate (PEC) and phenylethyl dimethylcaffeate (PEDMC) and tested them against the 3,2'-dimethyl-4-aminobiphenyl, (DMAB, a colon and mammary carcinogen)-induced mutagenicity in Salmonella typhimurium strains TA 98 and TA 100. Also, the effect of these agents on the growth of human colon adenocarcinoma, HT-29 cells and activities of ornithine decarboxylase (ODC) and protein tyrosine kinase (PTK) was studied. Mutagenicity was induced in Salmonella typhimurium strains TA 98 and TA 100 plus S9 activation using 5 and 10 micrograms DMAB and antimutagenic activities of 0-150 microM MC, 0-60 microM PEC and 0-80 microM PEDMC were determined. The results indicate that MC, PEC and PEDMC were not mutagenic in the Salmonella tester system. DMAB-induced mutagenicity was significantly inhibited with 150 microM MC, 40-60 microM PEC and 40-80 microM PEDMC in both tester systems. Treatment of HT-29 colon adenocarcinoma cells with > 150 microM MC, 30 microM PEC and 20 microM PEDMC significantly inhibited the cell growth and syntheses of RNA, DNA and protein. ODC and PTK activities were also inhibited in HT-29 cells treated with different concentrations of MC, PEC and PEDMC. These results demonstrate that caffeic acid esters which are present in Propolis possess chemopreventive properties when tested in short-term assay systems.

  2. Lipid Rafts Promote trans Fatty Acid-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

    PubMed

    Pan, Yao; Liu, Benxin; Deng, Zeyuan; Fan, Yawei; Li, Jing; Li, Hongyan

    2017-01-01

    The effects of two fatty acids, oleic acid (OLA) and elaidic acid (ELA) on normal human umbilical vein endothelial cells (HUVEC) and non-rafts HUVEC were investigated in this study. The expression levels of inflammatory cytokines (ICAM-1, VCAM-1 and IL-6) were analyzed. Western blot was used to analyze the expression levels of inflammation-related proteins (NF-κB, ERK1/2) and toll-like receptors 4 (TLR4). The results showed that the levels of nuclear translocation of NF-κB p65 and phosphorylated ERK1/2 were significantly decreased only in non-lipid rafts cells pretreated with trans fatty acid (TFA). The expression of TLR4 in the ELA-treated normal cells was higher than that in non-lipid rafts HUVEC. When the lipid rafts was destroyed by methyl-β-cyclodextrin, the levels of nuclear translocation of NF-κB p65, phosphorylated ERK1/2 and TLR4 were decreased significantly. Therefore, lipid rafts may be involved in TFA induced-inflammation in HUVEC through blocking the inflammatory signal pathway. Lipid rafts might be a platform for specific receptors such as TLR4 for TFA to activate the pro-inflammation on cell membranes.

  3. Dehydroabietic Acid Derivative QC2 Induces Oncosis in Hepatocellular Carcinoma Cells

    PubMed Central

    Zhang, Guang; Jiang, Chunping; Wang, Zhongxia; Chen, Weibo; Gu, Wen; Ding, Yitao

    2014-01-01

    Aim. Rosin, the traditional Chinese medicine, is reported to be able to inhibit skin cancer cell lines. In this report, we investigate the inhibitory effect against HCC cells of QC2, the derivative of rosin's main components dehydroabietic acid. Methods. MTT assay was used to determine the cytotoxicity of QC2. Morphological changes were observed by time-lapse microscopy and transmission electron microscopy and the cytoskeleton changes were observed by laser-scanning confocal microscopy. Cytomembrane integrity and organelles damage were confirmed by detection of the reactive oxygen (ROS), lactate dehydrogenase (LDH), and mitochondrial membrane potential (Δψm). The underlying mechanism was manifested by Western blotting. The oncotic cell death was further confirmed by detection of oncosis related protein calpain. Results. Swelling cell type and destroyed cytoskeleton were observed in QC2-treated HCC cells. Organelle damage was visualized by transmission electron microscopy. The detection of ROS accumulation, increased LDH release, and decreased ATP and Δψm confirmed the cell death. The oncotic related protein calpain was found to increase time-dependently in QC2-treated HCC cells, while its inhibitor PD150606 attenuated the cytotoxicity. Conclusions. Dehydroabietic acid derivative QC2 activated oncosis related protein calpain to induce the damage of cytomembrane and organelles which finally lead to oncosis in HCC cells. PMID:25110686

  4. Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid.

    PubMed

    Nitzsche, Astrid; Tokalov, Sergey V; Gutzeit, Herwig O; Ludwig-Müller, Jutta

    2004-05-19

    Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.

  5. Ouabain-Induced Signaling and Cell Survival in SK-N-SH Neuroblastoma Cells Differentiated by Retinoic Acid

    PubMed Central

    Akkuratov, Evgeny E.; Wu, Jian; Sowa, David; Shah, Zahoor A.; Liu, Lijun

    2015-01-01

    Ouabain stimulates activation of various signaling cascades such as protein kinase B (Akt) and Extracellular-signaling-regulated kinase 1/2 (ERK 1/2) in various cell lines. Retinoic acid (RA) is commonly used to induce neuroblastoma differentiation in cultures. Upon RA administration, human neuroblastoma cell line, SK-N-SH demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Here we report that ouabain-induced signaling is altered under the action of 1 μM RA in human neuroblastoma SK-N-SH cells. RA increased the expression of p110α subunit of phosphoinositide 3-kinase (PI3K), Akt and β1 subunit of Na+/K+-ATPase. Ouabain activated Akt and ERK 1/2 in differentiated SK-N-SH cells; this effect was not observed in non-differentiated SK-N-SH cells. Long-term incubation of non-differentiated SK-N-SH with 1 μM ouabain led to a decrease in the number of cells; this effect was reduced in differentiated SK-N-SH cells. Taken together, these results suggest that ouabain leads to cell death in neuroblastoma cells rather than neuronal cells due to the different response to ouabain manifested by activation of Akt and ERK 1/2. Highlights • RA increases the expression of p110α subunit of PI3K, Akt and β1 subunit of Na+/K+-ATPase • Ouabain induces activation of Akt and ERK 1/2 in differentiated SK-N-SH cells but not in non-differentiated cells • 1 μM ouabain leads to a decrease in the number of cells in non-differentiated SK-N-SH • Reduction of ouabain-induced cell death in differentiated SK-N-SH

  6. Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.

    PubMed

    Tian, Lin-Lin; Wang, Xue-Jun; Sun, Yu-Ning; Li, Chun-Rong; Xing, Ya-Ling; Zhao, Hai-Bao; Duan, Ming; Zhou, Zhe; Wang, Sheng-Qi

    2008-01-01

    Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.

  7. Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells.

    PubMed

    Pi, Jiang; Jin, Hua; Liu, Ruiying; Song, Bing; Wu, Qing; Liu, Li; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Cai, Jiye

    2013-02-01

    Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA-Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA-Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA-Se NPs in MCF-7 cells. The results indicated that the 70-nm FA-Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA-Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca(2)+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA-Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA-Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA-Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.

  8. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells.

    PubMed

    Wang, Li; Liu, Yuan; Li, Sen; Long, Zai-Yun; Wu, Ya-Min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and β-catenin. The results indicated that there were more β-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and β-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway.

  9. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis

    PubMed Central

    ZHAO, PENG; MAO, JUN-MIN; ZHANG, SHU-YUN; ZHOU, ZE-QUAN; TAN, YANG; ZHANG, YU

    2014-01-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a ‘chemopreventer’. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer. PMID:25009654

  10. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis.

    PubMed

    Zhao, Peng; Mao, Jun-Min; Zhang, Shu-Yun; Zhou, Ze-Quan; Tan, Yang; Zhang, Yu

    2014-08-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a 'chemopreventer'. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer.

  11. Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake

    PubMed Central

    Jakubowska, Monika A.; Gerasimenko, Julia V.; Gerasimenko, Oleg V.; Petersen, Ole H.

    2016-01-01

    Key points Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas.Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored.Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells.Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells.Bile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. Abstract Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca2+ signals and necrosis in acinar cells. However, bile acid‐elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3‐sulfate (TLC‐S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium–taurocholate cotransporting polypeptide (NTCP) indicate a Na

  12. Lysophosphatidic Acid Inhibits Apoptosis Induced by Cisplatin in Cervical Cancer Cells

    PubMed Central

    Sui, Yanxia; Yang, Ya; Wang, Ji; Li, Yi; Ma, Hongbing; Cai, Hui; Liu, Xiaoping; Zhang, Yong; Wang, Shufeng; Li, Zongfang; Zhang, Xiaozhi; Wang, Jiansheng; Liu, Rui; Yan, Yanli; Xue, Chaofan; Shi, Xiaowei; Tan, Li; Ren, Juan

    2015-01-01

    Cervical cancer is the second most common cause of cancer death in women worldwide. Lysophosphatidic acid (LPA) level has been found significantly increased in the serum of patients with ovarian, cervical, and colon cancers. LPA level in cervical cancer patients is significantly higher than in healthy controls. LPA receptors were found highly expressed in cervical cancer cells, suggesting LPA may play a role in the development of cervical cancer. The aim of this study is to investigate the effect of LPA on the apoptosis induced by cisplatin (DDP) in cervical cancer cell line and the underlying changes in signaling pathways. Our study found that cisplatin induced apoptosis of Hela cell through inhibiting expression of Bcl-2, upregulating the expression of Bax, Fas-L, and the enzyme activity of caspase-3 (p < 0.05); LPA significantly provided protection against the apoptosis induced by cisplatin by inhibiting the above alterations in apoptotic factor caused by cisplatin (p < 0.05). Moreover, PI3K/AKT pathway was found to be important for the LPA antiapoptosis effect, and administration of PI3K/AKT partially reversed the LPA-mediated protection against cisplatin-induced apoptosis (p < 0.05). These findings have shed new lights on the LPA bioactivity in cervical cancer cells and pointed to a possible sensitization scheme through combined administration of PI3K inhibitor and cisplatin for better treatment of cervical cancer patients, especially those with elevated LPA levels. PMID:26366416

  13. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    SciTech Connect

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  14. Ferulic acid prevents methylglyoxal-induced protein glycation, DNA damage, and apoptosis in pancreatic β-cells.

    PubMed

    Sompong, Weerachat; Cheng, Henrique; Adisakwattana, Sirichai

    2017-02-01

    Methylglyoxal (MG) can react with amino acids of proteins to induce protein glycation and consequently the formation of advanced glycation end-products (AGEs). Previous studies reported that ferulic acid (FA) prevented glucose-, fructose-, and ribose-induced protein glycation. In this study, FA (0.1-1 mM) inhibited MG-induced protein glycation and oxidative protein damage in bovine serum albumin (BSA). Furthermore, FA (0.0125-0.2 mM) protected against lysine/MG-mediated oxidative DNA damage, thereby inhibiting superoxide anion and hydroxyl radical generation during lysine and MG reaction. In addition, FA did not have the ability to trap MG. Finally, FA (0.1 mM) pretreatment attenuated MG-induced decrease in cell viability and prevented MG-induced cell apoptosis in pancreatic β-cells. The results suggest that FA is capable of protecting β-cells from MG-induced cell damage during diabetes.

  15. Lipotoxic effect of p21 on free fatty acid-induced steatosis in L02 cells.

    PubMed

    Wang, Jie-wei; Wan, Xing-yong; Zhu, Hua-tuo; Lu, Chao; Yu, Wei-lai; Yu, Chao-hui; Shen, Zhe; Li, You-ming

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) is increasingly regarded as a hepatic manifestation of metabolic syndrome. Though with high prevalence, the mechanism is poorly understood. This study aimed to investigate the effects of p21 on free fatty acid (FFA)-induced steatosis in L02 cells. We therefore analyzed the L02 cells with MG132 and siRNA treatment for different expression of p21 related to lipid accumulation and lipotoxicity. Cellular total lipid was stained by Oil Red O, while triglyceride content, cytotoxicity assays, lipid peroxidation markers and anti-oxidation levels were measured by enzymatic kits. Treatment with 1 mM FFA for 48 hr induced magnificent intracellular lipid accumulation and increased oxidative stress in p21 overload L02 cells compared to that in p21 knockdown L02 cells. By increasing oxidative stress and peroxidation, p21 accelerates FFA-induced lipotoxic effect in L02 cells and might provide information about potentially new targets for drug development and treatments of NAFLD.

  16. Free Fatty Acids Induce Autophagy and LOX-1 Upregulation in Cultured Aortic Vascular Smooth Muscle Cells.

    PubMed

    Cheng, Cheng-I; Lee, Yueh-Hong; Chen, Po-Han; Lin, Yu-Chun; Chou, Ming-Huei; Kao, Ying-Hsien

    2016-11-05

    Elevation of free fatty acids (FFAs) is known to affect microvascular function and contribute to obesity-associated insulin resistance, hypertension, and microangiopathy. Proliferative and synthetic vascular smooth muscle cells (VSMCs) increase intimal thickness and destabilize atheromatous plaques. This study aimed to investigate whether saturated palmitic acid (PA) and monounsaturated oleic acid (OA) modulate autophagy activity, cell proliferation, and vascular tissue remodeling in an aortic VSMC cell line. Exposure to PA and OA suppressed growth of VSMCs without apoptotic induction, but enhanced autophagy flux with elevation of Beclin-1, Atg5, and LC3I/II. Cotreatment with autophagy inhibitors potentiated the FFA-suppressed VSMC growth and showed differential actions of PA and OA in autophagy flux retardation. Both FFAs upregulated lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) but only OA increased LDL uptake by VSMCs. Mechanistically, FFAs induced hyperphosphorylation of Akt, ERK1/2, JNK1/2, and p38 MAPK. All pathways, except OA-activated PI3K/Akt cascade, were involved in the LOX-1 upregulation, whereas blockade of PI3K/Akt and MEK/ERK cascades ameliorated the FFA-induced growth suppression on VSMCs. Moreover, both FFAs exhibited tissue remodeling effect through increasing MMP-2 and MMP-9 expression and their gelatinolytic activities, whereas high-dose OA significantly suppressed collagen type I expression. Conversely, siRNA-mediated LOX-1 knockdown significantly attenuated the OA-induced tissue remodeling effects in VSMCs. In conclusion, OA and PA enhance autophagy flux, suppress aortic VSMC proliferation, and exhibit vascular remodeling effect, thereby leading to the loss of VSMCs and interstitial ECM in vascular walls and eventually the instability of atheromatous plaques. J. Cell. Biochem. 9999: 1-13, 2016. © 2016 Wiley Periodicals, Inc.

  17. Omega-3 free fatty acids inhibit tamoxifen-induced cell apoptosis.

    PubMed

    Wu, Shufan; Guo, Yang; Wu, Yikuan; Zhu, Shenglong; He, Zhao; Chen, Yong Q

    2015-04-03

    Fish oil, which contains omega-3 fatty acids mainly in the form of triglycerides, has benefits for reducing breast cancer risk, similar to tamoxifen action. However, it remains to be elucidated whether the combination of omega-3 free fatty acid (ω-3FFA) with tamoxifen leads to improved treatment in breast cancer. In this study, we observed that ω-3FFA induces MCF-7 cell apoptosis to suppress cell growth. The treatment of breast cancer cells with ω-3FFA attenuated tamoxifen-induced cell apoptosis. ω-3FFA and tamoxifen significantly increased Erk1/2 and Akt phosphorylation levels in a dose and time dependent manner. Compared to ω-3FFA alone, the combination of tamoxifen with ω-3FFA significantly increased Erk1/2 and Akt phosphorylation levels. Because Erk1/2 and Akt activation has been linked to tamoxifen-related anti-estrogen resistance in breast cancer patients, these results indicate that ω-3FFA may interfere with the effects of tamoxifen in the prevention of breast cancer risk.

  18. Lysophosphatidic acid rescues bone mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

    PubMed

    Wang, Xian-Yun; Fan, Xue-Song; Cai, Lin; Liu, Si; Cong, Xiang-Feng; Chen, Xi

    2015-03-01

    The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.

  19. Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

    PubMed Central

    Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; Patel, Neil K; Lu, Hua; Zeng, Shelya X; Wang, Guangdi; Zhang, Changde; You, Zongbing

    2014-01-01

    Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA’s effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, also named cIAP1), leading to activation of caspases 7 and 3 and turning on the downstream apoptotic events. MAA-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and CDK2 expression at the late time. MAA up-regulated p21 expression through inhibition of HDAC activities, independently of p53/p63/p73. These findings demonstrate that MAA suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which suggests that MAA could be used as a potential therapeutic drug for prostate cancer. PMID:25606576

  20. Chlorogenic acid prevents isoproterenol-induced DNA damage in vascular smooth muscle cells

    PubMed Central

    Wang, Jingshuai; Li, Jiyang; Liu, Jie; Xu, Mengjiao; Tong, Xiaowen; Wang, Jianjun

    2016-01-01

    Numerous clinical therapeutic agents have been identified as DNA damaging. The present study revealed that isoproterenol (Iso) resulted in DNA damage in vascular smooth muscle cells (VSMCs) and increased the levels of intracellular oxygen free radicals. Administration of chlorogenic acid (CGA) inhibited this effect. Pretreatment with CGA abrogated the increase in protein expression levels of γ-H2A histone family member X, phosphorylated ataxia telangiectasia mutated, phosphorylated Rad3-related protein, breast cancer 1 and C-terminal Src homologous kinase induced by Iso. In addition, the increase in levels of intracellular reactive oxygen species (ROS) induced by Iso was inhibited by CGA pretreatment in a dose-dependent manner. The results of the present study suggest that CGA may inhibit Iso-induced VSMC damage via the suppression of ROS generation. Therefore, CGA may be a novel agent for the treatment of vascular diseases. PMID:27634104

  1. Leucine deprivation inhibits proliferation and induces apoptosis of human breast cancer cells via fatty acid synthase

    PubMed Central

    Xiao, Fei; Wang, Chunxia; Yin, Hongkun; Yu, Junjie; Chen, Shanghai; Fang, Jing; Guo, Feifan

    2016-01-01

    Substantial studies on fatty acid synthase (FASN) have focused on its role in regulating lipid metabolism and researchers have a great interest in treating cancer with dietary manipulation of amino acids. In the current study, we found that leucine deprivation caused the FASN-dependent anticancer effect. Here we showed that leucine deprivation inhibited cell proliferation and induced apoptosis of MDA-MB-231 and MCF-7 breast cancer cells. In an in vivo tumor xenograft model, the leucine-free diet suppressed the growth of human breast cancer tumors and triggered widespread apoptosis of the cancer cells. Further study indicated that leucine deprivation decreased expression of lipogenic gene FASN in vitro and in vivo. Over-expression of FASN or supplementation of palmitic acid (the product of FASN action) blocked the effects of leucine deprivation on cell proliferation and apoptosis in vitro and in vivo. Moreover, leucine deprivation suppressed the FASN expression via regulating general control non-derepressible (GCN)2 and sterol regulatory element-binding protein 1C (SREBP1C). Taken together, our study represents proof of principle that anticancer effects can be obtained with strategies to deprive tumors of leucine via suppressing FASN expression, which provides important insights in prevention of breast cancer via metabolic intervention. PMID:27579768

  2. Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells

    PubMed Central

    Koskela, Ali; Reinisalo, Mika; Petrovski, Goran; Sinha, Debasish; Olmiere, Céline; Karjalainen, Reijo; Kaarniranta, Kai

    2016-01-01

    Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage. PMID:27187449

  3. Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells.

    PubMed

    Koskela, Ali; Reinisalo, Mika; Petrovski, Goran; Sinha, Debasish; Olmiere, Céline; Karjalainen, Reijo; Kaarniranta, Kai

    2016-05-11

    Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage.

  4. Valproic acid induces neuronal cell death through a novel calpain-dependent necroptosis pathway

    PubMed Central

    Bollino, Dominique; Balan, Irina; Aurelian, Laure

    2015-01-01

    A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor used to treat epilepsy and mood disorders, has HDAC-related and -unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain-dependent necroptosis pathway that initiates with downstream activation of c-Jun N-terminal kinase 1 (JNK1) and increased expression of receptor-interacting protein 1 (RIP-1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis-inducing-factor (AIF), mitochondrial release of Smac/DIABLO, and inhibition of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP). Coinciding with AIF nuclear translocation, VPA induces phosphorylation of the necroptosis-associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3-K/Akt survival pathways, allowing them to resist VPA-induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain-dependent necroptosis pathway that includes JNK1 activation and RIP-1 expression. PMID:25581256

  5. Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells To Induce Mucosal Tolerogenic Dendritic Cells.

    PubMed

    Goverse, Gera; Molenaar, Rosalie; Macia, Laurence; Tan, Jian; Erkelens, Martje N; Konijn, Tanja; Knippenberg, Marlene; Cook, Emma C L; Hanekamp, Diana; Veldhoen, Marc; Hartog, Anita; Roeselers, Guus; Mackay, Charles R; Mebius, Reina E

    2017-03-01

    The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.

  6. Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

    PubMed

    Stabellini, Giordano; Brugnoli, F; Calastrini, C; Vizzotto, L; Vertemati, M; Baroni, T; Caramelli, E; Marinucci, L; Pellati, A; Bertagnolo, V

    2004-01-01

    Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

  7. Retinoic-acid-orphan-receptor-C inhibition suppresses Th17 cells and induces thymic aberrations

    PubMed Central

    Guntermann, Christine; Piaia, Alessandro; Hamel, Marie-Laure; Theil, Diethilde; Rubic-Schneider, Tina; del Rio-Espinola, Alberto; Dong, Linda; Billich, Andreas; Kaupmann, Klemens; Dawson, Janet; Hoegenauer, Klemens; Orain, David; Hintermann, Samuel; Stringer, Rowan; Patel, Dhavalkumar D.; Doelemeyer, Arno; Deurinck, Mark

    2017-01-01

    Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic Rorc deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as Bcl2l1 and BCL2L1 mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in Rorc-deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with neoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in Rorc-deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk. PMID:28289717

  8. Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

    PubMed Central

    Huang, Wen-Shih; Yang, Jen-Tsung; Lu, Chien-Chang; Chang, Shun-Fu; Chen, Cheng-Nan; Su, Yu-Ping; Lee, Ko-Chao

    2015-01-01

    A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC). Hence, resistin may play a role in CRC development. Fulvic acid (FA), a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative) and SW-48 (p53-positive) CRC cells and human umbilical vein endothelial cells (HUVECs) were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin. PMID:26690142

  9. Nitro-Arachidonic Acid Prevents Angiotensin II-Induced Mitochondrial Dysfunction in a Cell Line of Kidney Proximal Tubular Cells.

    PubMed

    Sánchez-Calvo, Beatriz; Cassina, Adriana; Rios, Natalia; Peluffo, Gonzalo; Boggia, José; Radi, Rafael; Rubbo, Homero; Trostchansky, Andres

    2016-01-01

    Nitro-arachidonic acid (NO2-AA) is a cell signaling nitroalkene that exerts anti-inflammatory activities during macrophage activation. While angiotensin II (ANG II) produces an increase in reactive oxygen species (ROS) production and mitochondrial dysfunction in renal tubular cells, little is known regarding the potential protective effects of NO2-AA in ANG II-mediated kidney injury. As such, this study examines the impact of NO2-AA on ANG II-induced mitochondrial dysfunction in an immortalized renal proximal tubule cell line (HK-2 cells). Treatment of HK-2 cells with ANG II increases the production of superoxide (O2●-), nitric oxide (●NO), inducible nitric oxide synthase (NOS2) expression, peroxynitrite (ONOO-) and mitochondrial dysfunction. Using high-resolution respirometry, it was observed that the presence of NO2-AA prevented ANG II-mediated mitochondrial dysfunction. Attempting to address mechanism, we treated isolated rat kidney mitochondria with ONOO-, a key mediator of ANG II-induced mitochondrial damage, in the presence or absence of NO2-AA. Whereas the activity of succinate dehydrogenase (SDH) and ATP synthase (ATPase) were diminished upon exposure to ONOO-, they were restored by pre-incubating the mitochondria with NO2-AA. Moreover, NO2-AA prevents oxidation and nitration of mitochondrial proteins. Combined, these data demonstrate that ANG II-mediated oxidative damage and mitochondrial dysfunction is abrogated by NO2-AA, identifying this compound as a promising pharmacological tool to prevent ANG II-induced renal disease.

  10. Sonic hedgehog and retinoic Acid induce bone marrow-derived stem cells to differentiate into glutamatergic neural cells.

    PubMed

    Yu, Zhenhai; Wu, Shixing; Liu, Zhen; Lin, Haiyan; Chen, Lei; Yuan, Xinli; Zhang, Zhiying; Liu, Fang; Zhang, Chuansen

    2015-01-01

    Studies have showed that transplanted stem cells in the inner ear won't regenerate to replace the damaged sensory hair cells. They can spontaneously differentiate into mesenchymal cells and fibrocytes in the damaged inner ear. Only mature sensory cells of MSCs-derived possess the great potency for cell transplantation in the treatment of sensorineural hearing loss. So, we try to establish an efficient generation of the glutamatergic sensory neural phenotype for the cell transplantation of the hearing loss. We isolated MSCs from femoral and tibial bones according to their adherence to culture dishes. After purification, proliferation, and passaged, cells became homogeneous in appearance, showing more uniformity and grew in a monolayer with a typical spindle-shape morphology. The cell surface markers were assessed using FACS to characterize the isolated cells. For neural induction to harvest the glutamatergic sensory neurons, passage 3 MSCs were incubated with preinduced medium for 24 hr, and neural-induced medium for an additional 14 days. The cells exhibit a typical neural shape. RT-PCR analysis indicated that the mRNA levels of the neural cell marker nestin, Tau, MAP-2, β-tubulin III, GluR-3, and GluR-4 were higher compared with primary MSCs. Immunohistochemistry and western-blotting proofed that nestin, MAP-2, β-tubulin III, and GluR-4 proteins indeed exhibit their expression difference in the induced cells compared to the MSCs. We show an efficient protocol by the combined applications of Sonic Hedgehog (Shh) and Retinoic Acid (RA) to induce MSCs to differentiate into the glutamatergic sensory neuron which were identified from the morphological, biochemical, and molecular characteristics.

  11. The Acid Growth Theory of auxin-induced cell elongation is alive and well

    NASA Technical Reports Server (NTRS)

    Rayle, D. L.; Cleland, R. E.

    1992-01-01

    Plant cells elongate irreversibly only when load-bearing bonds in the walls are cleaved. Auxin causes the elongation of stem and coleoptile cells by promoting wall loosening via cleavage of these bonds. This process may be coupled with the intercalation of new cell wall polymers. Because the primary site of auxin action appears to be the plasma membrane or some intracellular site, and wall loosening is extracellular, there must be communication between the protoplast and the wall. Some "wall-loosening factor" must be exported from auxin-impacted cells, which sets into motion the wall loosening events. About 20 years ago, it was suggested that the wall-loosening factor is hydrogen ions. This idea and subsequent supporting data gave rise to the Acid Growth Theory, which states that when exposed to auxin, susceptible cells excrete protons into the wall (apoplast) at an enhanced rate, resulting in a decrease in apoplastic pH. The lowered wall pH then activates wall-loosening processes, the precise nature of which is unknown. Because exogenous acid causes a transient (1-4 h) increase in growth rate, auxin must also mediate events in addition to wall acidification for growth to continue for an extended period of time. These events may include osmoregulation, cell wall synthesis, and maintenance of the capacity of walls to undergo acid-induced wall loosening. At present, we do not know if these phenomena are tightly coupled to wall acidification or if they are the products of multiple independent signal transduction pathways.

  12. Myoinositol Attenuates the Cell Loss and Biochemical Changes Induced by Kainic Acid Status Epilepticus

    PubMed Central

    Kikvidze, Marina

    2016-01-01

    Identification of compounds preventing or modifying the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action are of great importance. We have previously shown that myoinositol (MI) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (KA) induced status epilepticus (SE). However in these studies we have not detected any effects of MI on the first day after SE. In the present study we broadened our research and focused on other molecular and morphological changes at the early stages of SE induced by KA and effects of MI treatment on these changes. The increase in the amount of voltage-dependent anionic channel-1 (VDAC-1), cofilin, and caspase-3 activity was observed in the hippocampus of KA treated rats. Administration of MI 4 hours later after KA treatment abolishes these changes, whereas diazepam treatment by the same time schedule has no significant influence. The number of neuronal cells in CA1 and CA3 subfields of hippocampus is decreased after KA induced SE and MI posttreatment significantly attenuates this reduction. No significant changes are observed in the neocortex. Obtained results indicate that MI posttreatment after KA induced SE could successfully target the biochemical processes involved in apoptosis, reduces cell loss, and can be successfully used in the future for translational research. PMID:27642592

  13. Purified eicosapentaenoic acid induces prolonged survival of cardiac allografts and generates regulatory T cells.

    PubMed

    Iwami, D; Zhang, Q; Aramaki, O; Nonomura, K; Shirasugi, N; Niimi, M

    2009-06-01

    Fish oil, which is rich in eicosapentaenoic acid (EPA), has been found to have immunomodulatory effects. We examined whether administration of purified EPA affected survival of fully mismatched murine cardiac allografts. Hearts from C57BL/10 (H-2(b)) mice were transplanted into CBA (H-2(k)) recipients treated with one intraperitoneal dose of purified EPA the day of transplantation. Untreated CBA recipients and recipients given 0.1 g/kg of EPA rejected C57BL/10 hearts (median survival time [MST], 8 and 13 days, respectively). With a 1.0 g/kg dose of EPA, graft survival was markedly prolonged (MST >100 days). To determine whether regulatory cells were generated, naïve mice (secondary recipients) underwent adoptive transfer of splenocytes from EPA-treated primary recipients and cardiac allograft transplantation. Adoptive transfer of whole, CD4(+) and CD4(+)CD25(+) splenocytes from EPA-treated recipients induced indefinite survival in secondary recipients. Flow cytometry showed that the CD4(+)CD25(+) cells were Foxp3(+). In reverse transcriptase-polymerase chain reaction (RT-PCR) studies, the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA was upregulated by EPA treatment. A PPARgamma antagonist abrogated the prolongation of graft survival induced by EPA treatment (MST, 13 days). Thus, in our model, purified EPA induced prolonged survival of fully mismatched cardiac allografts and generated regulatory T cells dependent on PPARgamma activation.

  14. Trans Fatty Acids Induce Vascular Inflammation and Reduce Vascular Nitric Oxide Production in Endothelial Cells

    PubMed Central

    Iwata, Naomi G.; Pham, Matilda; Rizzo, Norma O.; Cheng, Andrew M.; Maloney, Ezekiel; Kim, Francis

    2011-01-01

    Intake of trans fatty acids (TFA), which are consumed by eating foods made from partially hydrogenated vegetable oils, is associated with a higher risk of cardiovascular disease. This relation can be explained by many factors including TFA's negative effect on endothelial function and reduced nitric oxide (NO) bioavailability. In this study we investigated the effects of three different TFA (2 common isomers of C18 found in partially hydrogenated vegetable oil and a C18 isomer found from ruminant-derived—dairy products and meat) on endothelial NF-κB activation and nitric oxide (NO) production. Human endothelial cells were treated with increasing concentrations of Elaidic (trans-C18:1 (9 trans)), Linoelaidic (trans-C18:2 (9 trans, 12 trans)), and Transvaccenic (trans-C18:1 (11 trans)) for 3 h. Both Elaidic and Linoelaidic acids were associated with increasing NF-κB activation as measured by IL-6 levels and phosphorylation of IκBα, and impairment of endothelial insulin signaling and NO production, whereas Transvaccenic acid was not associated with these responses. We also measured superoxide production, which has been hypothesized to be necessary in fatty acid-dependent activation of NF-κB. Both Elaidic acid and Linoelaidic acid are associated with increased superoxide production, whereas Transvaccenic acid (which did not induce inflammatory responses) did not increase superoxide production. We observed differential activation of endothelial superoxide production, NF-κB activation, and reduction in NO production by different C18 isomers suggesting that the location and number of trans double bonds effect endothelial NF-κB activation. PMID:22216328

  15. Nerve growth factor protects against palmitic acid-induced injury in retinal ganglion cells

    PubMed Central

    Yan, Pan-shi; Tang, Shu; Zhang, Hai-feng; Guo, Yuan-yuan; Zeng, Zhi-wen; Wen, Qiang

    2016-01-01

    Accumulating evidence supports an important role for nerve growth factor (NGF) in diabetic retinopathy. We hypothesized that NGF has a protective effect on rat retinal ganglion RGC-5 cells injured by palmitic acid (PA), a metabolic factor implicated in the development of diabetes and its complications. Our results show that PA exposure caused apoptosis of RGC-5 cells, while NGF protected against PA insult in a concentration-dependent manner. Additionally, NGF significantly attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in RGC-5 cells. Pathway inhibitor tests showed that the protective effect of NGF was completely reversed by LY294002 (PI3K inhibitor), Akt VIII inhibitor, and PD98059 (ERK1/2 inhibitor). Western blot analysis revealed that NGF induced the phosphorylation of Akt/FoxO1 and ERK1/2 and reversed the PA-evoked reduction in the levels of these proteins. These results indicate that NGF protects RGC-5 cells against PA-induced injury through anti-oxidation and inhibition of apoptosis by modulation of the PI3K/Akt and ERK1/2 signaling pathways. PMID:28123432

  16. Salvianolic acid Y: a new protector of PC12 cells against hydrogen peroxide-induced injury from Salvia officinalis.

    PubMed

    Gong, Jun; Ju, Aichun; Zhou, Dazheng; Li, Dekun; Zhou, Wei; Geng, Wanli; Li, Bing; Li, Li; Liu, Yanjie; He, Ying; Song, Meizhen; Wang, Yunhua; Ye, Zhengliang; Lin, Ruichao

    2015-01-06

    Salvianolic acid Y (TSL 1), a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC) and circular dichroism (CD) experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1) was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1) protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%).

  17. Deoxycholic Acid Could Induce Apoptosis and Trigger Gastric Carcinogenesis on Gastric Epithelial Cells by Quantitative Proteomic Analysis

    PubMed Central

    Wei, Ying; Zhang, Jing; Wang, Ye

    2016-01-01

    Background. Pathologic duodenogastric reflux can induce or aggravate gastritis because of the presence of bile acids. Bile reflux has been generally considered to be associated with intestinal metaplasia and gastric cancer. However, the pathogenic mechanisms of the effects of bile acids on gastric mucosa are still unknown. Methods. To explore the mechanisms by which bile acids induce gastric mucosal lesions, we examined cell apoptosis in the gastric epithelial cell line GES-1 and investigated the changes in protein profiles of GES-1 cells in response to a bile acid deoxycholic acid using a proteomics approach. Changes in the profiles of the differently expressed proteins were analyzed using the DAVID and STRING programs. Results. We found apoptosis was significantly induced in GES-1 cells by deoxycholic acid. Using liquid chromatographic/tandem mass spectrometric (LC-MS/MS) methods, 134 upregulated proteins and 214 downregulated proteins were identified in the bile acid treated GES-1 cells. Bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. Conclusion. These findings may improve the understanding of the molecular mechanisms underlying the pathogenicity of bile acids on gastric mucosa. PMID:28070185

  18. Deoxycholic Acid Could Induce Apoptosis and Trigger Gastric Carcinogenesis on Gastric Epithelial Cells by Quantitative Proteomic Analysis.

    PubMed

    Shi, Yanyan; Wei, Ying; Zhang, Ting; Zhang, Jing; Wang, Ye; Ding, Shigang

    2016-01-01

    Background. Pathologic duodenogastric reflux can induce or aggravate gastritis because of the presence of bile acids. Bile reflux has been generally considered to be associated with intestinal metaplasia and gastric cancer. However, the pathogenic mechanisms of the effects of bile acids on gastric mucosa are still unknown. Methods. To explore the mechanisms by which bile acids induce gastric mucosal lesions, we examined cell apoptosis in the gastric epithelial cell line GES-1 and investigated the changes in protein profiles of GES-1 cells in response to a bile acid deoxycholic acid using a proteomics approach. Changes in the profiles of the differently expressed proteins were analyzed using the DAVID and STRING programs. Results. We found apoptosis was significantly induced in GES-1 cells by deoxycholic acid. Using liquid chromatographic/tandem mass spectrometric (LC-MS/MS) methods, 134 upregulated proteins and 214 downregulated proteins were identified in the bile acid treated GES-1 cells. Bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. Conclusion. These findings may improve the understanding of the molecular mechanisms underlying the pathogenicity of bile acids on gastric mucosa.

  19. Jacaric acid and its octadecatrienoic acid geoisomers induce apoptosis selectively in cancerous human prostate cells: a mechanistic and 3-D structure-activity study.

    PubMed

    Gasmi, Jihane; Thomas Sanderson, J

    2013-06-15

    Plant-derived non-essential fatty acids are important dietary nutrients, and some are purported to have chemopreventive properties against various cancers, including that of the prostate. In this study, we determined the ability of seven dietary C-18 fatty acids to cause cytotoxicity and induce apoptosis in various types of human prostate cancer cells. These fatty acids included jacaric and punicic acid found in jacaranda and pomegranate seed oil, respectively, three octadecatrienoic geometric isomers (alpha- and beta-calendic and catalpic acid) and two mono-unsaturated C-18 fatty acids (trans- and cis-vaccenic acid). Jacaric acid and four of its octadecatrienoic geoisomers selectively induced apoptosis in hormone-dependent (LNCaP) and -independent (PC-3) human prostate cancer cells, whilst not affecting the viability of normal human prostate epithelial cells (RWPE-1). Jacaric acid induced concentration- and time-depedent LNCaP cell death through activation of intrinsic and extrinsic apoptotic pathways resulting in cleavage of PARP-1, modulation of pro- and antiapoptotic Bcl-2 family of proteins and increased cleavage of caspase-3, -8 and -9. Moreover, activation of a cell death-inducing signalling cascade involving death receptor 5 was observed. Jacaric acid induced apoptosis in PC-3 cells by activation of the intrinsic pathway only. The spatial conformation cis, trans, cis of jacaric and punicic acid was shown to play a key role in the increased potency and efficacy of these two fatty acids in comparison to the five other C-18 fatty acids tested. Three-dimensional conformational analysis using the PubChem Database (http://pubchem.ncbi.nlm.nih.gov) showed that the cytotoxic potency of the C-18 fatty acids was related to their degree of conformational similarity to our cytotoxic reference compound, punicic acid, based on optimized shape (ST) and feature (CT) similarity scores, with jacaric acid being most 'biosimilar' (ST(ST-opt)=0.81; CT(CT-opt)=0.45). This 3-D

  20. Oleanolic acid inhibits proliferation and induces apoptosis in NB4 cells by targeting PML/RARα

    PubMed

    Li, Hongmei; He, Ning; Li, Xueyan; Zhou, Li; Zhao, Mingyu; Jiang, Hairui; Zhang, Xiaojie

    2013-10-01

    Oleanolic acid (OA), a naturally occurring pentacyclic triterpenoid contained in a variety of plant species, exhibits broad biological properties, including anticancer effects. Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia. APL has a unique and specific chromosomal aberration, t(15;17), which results in the formation of a fusion gene and protein PML/RARα, which is not only necessary for the diagnosis of APL, but is also critical for APL pathogenesis. In the present study, the cytotoxic effect of OA on NB4 cells was investigated. Cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of bax and bcl-2 mRNA were determined by quantitative PCR. Apoptosis was analyzed using DNA fragment analysis and cell cycle distributions were analyzed by flow cytometry. The activity of caspase-3 and caspase-9 was determined by colorimetric assays. The expression of the PML/RARα fusion protein was analyzed by western blotting. The MTT assay showed that OA inhibited the proliferation of the NB4 cells. The expression levels of pro-apoptotic bax mRNA were increased and the levels of anti-apoptotic bcl-2 mRNA were decreased following the treatment of the NB4 cells with OA at 80 μmol/l. Treatment of the NB4 cells with OA at 80 μmol/l induced apoptosis and G1 phase arrest, while caspase-9 and caspase-3 activity was significantly increased. Furthermore, the expression of the PML/RARα fusion protein was decreased. Together, these data suggest that OA exerts a cytotoxic effect that inhibits proliferation and induces apoptosis in NB4 cells by targeting PML/RARα, making it a potent therapeutic agent against leukemia.

  1. Ascorbic Acid and a Cytostatic Inhibitor of Glycolysis Synergistically Induce Apoptosis in Non-Small Cell Lung Cancer Cells

    PubMed Central

    Vuyyuri, Saleha B.; Rinkinen, Jacob; Worden, Erin; Shim, Hyekyung; Lee, Sukchan; Davis, Keith R.

    2013-01-01

    Ascorbic acid (AA) exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small cell lung cancer (NSCLC) cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a <10% loss of viability in the lung epithelial cell line. Combinations of AA and 3-PO synergistically enhanced cell death in all NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone. A synergistic interaction was not observed in combination treatments of lung epithelial cells and combination treatments that caused a complete loss of viability in NSCLC cells had modest effects on normal lung cell viability and reactive oxygen species (ROS) levels. Combination treatments induced dramatically higher ROS levels compared to treatment with AA and 3-PO alone in NSCLC cells and combination-induced cell death was inhibited by addition of catalase to the medium. Analyses of DNA fragmentation, poly (ADP-ribose) polymerase cleavage, annexin V-binding, and caspase activity demonstrated that AA-induced cell death is caused via the activation of apoptosis and that the combination treatments caused a synergistic induction of apoptosis. These results demonstrate the effectiveness of AA against NSCLC cells and that combinations of AA with 3-PO synergistically induce apoptosis via a ROS-dependent mechanism. These results support further evaluation of pharmacologic concentrations of AA as an adjuvant treatment for NSCLC and that combination of AA with glycolysis

  2. Folic Acid Protected Neural Cells Against Aluminum-Maltolate-Induced Apoptosis by Preventing miR-19 Downregulation.

    PubMed

    Zhu, Mingming; Li, Bingfei; Ma, Xiao; Huang, Cong; Wu, Rui; Zhu, Weiwei; Li, Xiaoting; Liang, Zhaofeng; Deng, Feifei; Zhu, Jianyun; Xie, Wei; Yang, Xue; Jiang, Ye; Wang, Shijia; Wu, Jieshu; Geng, Shanshan; Xie, Chunfeng; Zhong, Caiyun; Liu, Haiyan

    2016-08-01

    Aluminum (Al)-induced apoptosis is considered as the major cause of its neurotoxicity. Folic acid possesses neuroprotective function by preventing neural cell apoptosis. microRNAs (miRNAs) are important regulators of gene expression participating in cellular processes. As a key component of the miR-17-92 cluster, miR-19 is implicated in regulating apoptotic process, while its role in the neuroprotective effect of folic acid has not been investigated. The present study aimed to investigate the potential involvement and function of miR-19 in the protective action of folic acid against Al-induced neural cell apoptosis. Human SH-SY5Y cells were treated with Al-maltolate (Al-malt) in the presence or absence of folic acid. Results showed that Al-malt-induced apoptosis of SH-SY5Y cells was effectively prevented by folic acid. Al-malt suppressed the expression of miR-19a/19b, along with alterations of miR-19 related apoptotic proteins including PTEN, p-AKT, p53, Bax, Bcl-2, caspase 9 and caspase 3; and these effects were ameliorated by folic acid. miR-19 inhibitor alone induced apoptosis of SH-SY5Y cells. Combination treatment of folic acid and miR-19 inhibitor diminished the neuroprotective effect of folic acid. These findings demonstrated that folic acid protected neuronal cells against Al-malt-induced apoptosis by preventing the downregulation of miR-19 and modulation of miR-19 related downstream PTEN/AKT/p53 pathway.

  3. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation

    SciTech Connect

    Kauss, M. Ariel; Reiterer, Gudrun; Bunaciu, Rodica P.; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G{sub 1} to S to G{sub 2}/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G{sub 0} cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  4. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation.

    PubMed

    Kauss, M Ariel; Reiterer, Gudrun; Bunaciu, Rodica P; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G(1) to S to G(2)/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G(0) cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  5. Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation

    SciTech Connect

    Svensson Holm, Ann-Charlotte B.; Bengtsson, Torbjoern; Grenegard, Magnus; Lindstroem, Eva G.

    2012-03-10

    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.

  6. Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells.

    PubMed

    Lanteri, M Luciana; Lamattina, Lorenzo; Laxalt, Ana M

    2011-10-01

    The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant-pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.

  7. Butoxyacetic acid-induced hemolysis of rat red blood cells: effect of external osmolarity and cations.

    PubMed

    Udden, M M; Patton, C S

    2005-03-28

    Hemolysis is the principal toxicity of acute exposure to ethylene glycol monobutyl ether (EGBE) in rats. EGBE itself is not an active hemolytic agent, but its metabolite, butoxyacetic acid (BAA) formed as a result of dehydrogenase activity is a potent hemolysin. Here we address the role of osmolarity and cation composition of the suspending buffers in the mechanism of BAA-induced hemolysis of rat red blood cells in vitro. Rat erythrocytes were protected from BAA-induced cell swelling and hemolysis by the addition of sucrose to the suspending media. Hemolysis and cell swelling were also reduced by replacing external sodium with potassium. When calcium was not present in the suspending medium or when chelated by EGTA, hemolysis was increased after 2 h incubation with 1 mM or 2 mM BAA. Addition of as little as 0.05 mM CaCl(2) reduced hemolysis significantly while the addition of MgCl(2) had no effect. The dose-response relationship between BAA concentration and hemolysis determined in the presence or absence of calcium showed an increased effect of BAA in the absence of calcium. BAA-induced spherocytosis and cell fragmentation were more pronounced in the absence of calcium. The time course of BAA-induced hemolysis in the presence and absence of calcium demonstrated that the effect of calcium is to delay the onset of hemolysis. Increased intracellular calcium as a result of exposure to BAA was verified by atomic absorption spectroscopy. Charybdotoxin, an inhibitor of the calcium activated potassium channel, blocked the protective effect of calcium suggesting that the delay of onset of hemolysis in the presence of calcium is due to potassium loss caused by this channel. We conclude that the mode of action of BAA is to cause a colloid osmotic lysis of the rat red blood cell. Hemolysis requires external sodium and is associated with calcium uptake. Calcium appears to delay the onset of hemolysis. We speculate that: (1) BAA causes sodium and calcium to enter the cell; (2

  8. Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death.

    PubMed

    Lu, Yapeng; Liu, Siyuan; Wang, Ying; Wang, Dang; Gao, Jing; Zhu, Li

    2016-09-05

    Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca(2+) release from Ca(2+)-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca(2+)-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca(2+) release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells.

  9. Protective effect of boric acid on lead- and cadmium-induced genotoxicity in V79 cells.

    PubMed

    Ustündağ, Aylin; Behm, Claudia; Föllmann, Wolfram; Duydu, Yalçin; Degen, Gisela H

    2014-06-01

    The toxic heavy metals cadmium (Cd) and lead (Pb) are important environmental pollutants which can cause serious damage to human health. As the metal ions (Cd(2+) and Pb(2+)) accumulate in the organism, there is special concern regarding chronic toxicity and damage to the genetic material. Metal-induced genotoxicity has been attributed to indirect mechanisms, such as induction of oxidative stress and interference with DNA repair. Boron is a naturally occurring element and considered to be an essential micronutrient, although the cellular activities of boron compounds remain largely unexplored. The present study has been conducted to evaluate potential protective effects of boric acid (BA) against genotoxicity induced by cadmium chloride (CdCl2) and lead chloride (PbCl2) in V79 cell cultures. Cytotoxicity assays (neutral red uptake and cell titer blue assay) served to determine suitable concentrations for subsequent genotoxicity assays. Chromosomal damage and DNA strand breaks were assessed by micronucleus tests and comet assays. Both PbCl2 and CdCl2 (at 3, 5 and 10 µM) were shown to induce concentration-dependent increases in micronucleus frequencies and DNA strand breaks in V79 cells. BA itself was not cytotoxic (up to 300 µM) and showed no genotoxic effects. Pretreatment of cells with low levels of BA (2.5 and 10 µM) was found to strongly reduce the genotoxic effects of the tested metals. Based on the findings of this in vitro study, it can be suggested that boron provides an efficient protection against the induction of DNA strand breaks and micronuclei by lead and cadmium. Further studies on the underlying mechanisms for the protective effect of boron are needed.

  10. Apoptosis- and differentiation-inducing activities of jacaric acid, a conjugated linolenic acid isomer, on human eosinophilic leukemia EoL-1 cells.

    PubMed

    Liu, Wai-Nam; Leung, Kwok-Nam

    2014-11-01

    Conjugated linolenic acids (CLNAs) are a group of naturally occurring positional and geometrical isomers of the C18 polyunsaturated essential fatty acid, linolenic acid (LNA), with three conjugated double bonds (C18:3). Although previous research has demonstrated the growth-inhibitory effects of CLNA on a wide variety of cancer cell lines in vitro, their action mechanisms and therapeutic potential on human myeloid leukemia cells remain poorly understood. In the present study, we found that jacaric acid (8Z,10E,12Z-octadecatrienoic acid), a CLNA isomer which is present in jacaranda seed oil, inhibited the in vitro growth of human eosinophilic leukemia EoL-1 cells in a time- and concentration-dependent manner. Mechanistic studies showed that jacaric acid triggered cell cycle arrest of EoL-1 cells at the G0/G1 phase and induced apoptosis of the EoL-1 cells, as measured by the Cell Death Detection ELISAPLUS kit, Annexin V assay and JC-1 dye staining. Notably, the jacaric acid-treated EoL-1 cells also underwent differentiation as revealed by morphological and phenotypic analysis. Collectively, our results demonstrated the capability of jacaric acid to inhibit the growth of EoL-1 cells in vitro through triggering cell cycle arrest and by inducing apoptosis and differentiation of the leukemia cells. Therefore, jacaric acid might be developed as a potential candidate for the treatment of certain forms of myeloid leukemia with minimal toxicity and few side effects.

  11. The Histone Deacetylase Inhibitor Vaproic Acid Induces Cell Growth Arrest in Hepatocellular Carcinoma Cells via Suppressing Notch Signaling

    PubMed Central

    Sun, Guangchun; Mackey, Lily V.; Coy, David H.; Yu, Cui-Yun; Sun, Lichun

    2015-01-01

    Hepatocellular carcinoma (HCC) is a type of malignant cancer. Notch signaling is aberrantly expressed in HCC tissues with more evidence showing that this signaling plays a critical role in HCCs. In the present study, we investigate the effects of the anti-convulsant drug valproic acid (VPA) in HCC cells and its involvement in modulating Notch signaling. We found that VPA, acting as a histone deacetylase (HDAC) inhibitor, induced a decrease in HDAC4 and an increase in acetylated histone 4 (AcH4) and suppressed HCC cell growth. VPA also induced down-regulation of Notch signaling via suppressing the expression of Notch1 and its target gene HES1, with an increase of tumor suppressor p21 and p63. Furthermore, Notch1 activation via overexpressing Notch1 active form ICN1 induced HCC cell proliferation and anti-apoptosis, indicating Notch signaling played an oncogenic role in HCC cells. Meanwhile, VPA could reverse Notch1-induced increase of cell proliferation. Interestingly, VPA was also observed to stimulate the expression of G protein-coupled somatostatin receptor type 2 (SSTR2) that has been used in receptor-targeting therapies. This discovery supports a combination therapy of VPA with the SSTR2-targeting agents. Our in vitro assay did show that the combination of VPA and the peptide-drug conjugate camptothecin-somatostatin (CPT-SST) displayed more potent anti-proliferative effects on HCC cells than did each alone. VPA may be a potential drug candidate in the development of anti-HCC drugs via targeting Notch signaling, especially in combination with receptor-targeting cytotoxic agents. PMID:26366213

  12. Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

    PubMed Central

    Durachko, Daniel M.; Cosgrove, Daniel J.

    2009-01-01

    Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (< 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls. To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the

  13. Ursodeoxycholic acid (UDCA) can inhibit deoxycholic acid (DCA)-induced apoptosis via modulation of EGFR/Raf-1/ERK signaling in human colon cancer cells.

    PubMed

    Im, Eunok; Martinez, Jesse D

    2004-02-01

    Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is known as a cytoprotective agent. UDCA prevents apoptosis induced by a variety of stress stimuli including cytotoxic bile acids such as deoxycholic acid (DCA). Here we examined the molecular mechanism by which UDCA can antagonize DCA-induced apoptosis in human colon cancer cells. UDCA pretreatment decreases the number of apoptotic cells caused by exposure to DCA and UDCA. Further studies of the signaling pathway showed that UDCA pretreatment suppressed DNA binding activity of activator protein-1 and this was accompanied by downregulation of both extracellular signal-regulated kinase (ERK) and Raf-1 kinase activities stimulated by exposure to DCA. DCA was also found to activate epidermal growth factor receptor (EGFR) activity and UDCA inhibited this. Collectively, these findings suggest that the inhibitory effect of UDCA in DCA-induced apoptosis is partly mediated by modulation of EGFR/Raf-1/ERK signaling.

  14. Rutin inhibits oleic acid induced lipid accumulation via reducing lipogenesis and oxidative stress in hepatocarcinoma cells.

    PubMed

    Wu, Cheng-Hsun; Lin, Ming-Cheng; Wang, Hsueh-Chun; Yang, Mon-Yuan; Jou, Ming-Jia; Wang, Chau-Jong

    2011-03-01

    Excessive lipid accumulation within liver has been proposed to cause obesity, hyperlipidemia, diabetes, and fatty liver disease. Rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, has various biological functions, including antioxidant, anti-inflammatory, and anticancer effects. However, a hypolipidemic effect of rutin on fatty liver disease has not been reported. In this study, we examined the effect of rutin on reducing lipid accumulation in hepatic cells. Hepatocytes were treated with oleic acid (OA) containing with or without rutin to observe the lipid accumulation by Nile red stain. The result showed rutin suppressed OA-induced lipid accumulation and increased adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activity in hepatocytes. The expression of critical molecule involved in lipid synthesis, sterol regulatory element binding proteins-1 (SREBP-1), was attenuated in rutin-treated cells. Moreover, long-term incubation of rutin inhibited the transcriptions of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), glycerol-3-phosphate acyltransferase (GPAT), fatty acid synthase (FAS), and acetyl-coenzyme carboxylase (ACC). Besides, we also found out the antioxidative effect of rutin by increasing the expression of peroxisome proliferator-activated receptor (PPAR)-α and antioxidative enzymes. Taken together, our findings suggest rutin could attenuate lipid accumulation by decreasing lipogenesis and oxidative stress in hepatocyte.

  15. Modulation of chemotherapy-induced cytotoxicity in SH-SY5Y neuroblastoma cells by caffeine and chlorogenic acid.

    PubMed

    Hall, Susan; Anoopkumar-Dukie, Shailendra; Grant, Gary D; Desbrow, Ben; Lai, Richard; Arora, Devinder; Hong, Yinna

    2017-03-06

    Chemotherapy is an important treatment modality for malignancy but is limited by significant toxicity and it susceptibility to numerous drug interactions. While the interacting effects with medications are well known, there is limited evidence on the interaction with commonly consumed food and natural products. The aim of this study was to evaluate the bioactive constituents of coffee (caffeine and chlorogenic acid) on the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in vitro. Pretreatment with caffeine (100 nM and 10 μM) sensitized SH-SY5Y cells to doxorubicin-induced toxicity and increased apoptosis and sensitized PC3 cells to gemcitabine-induced toxicity. Pretreatment with 10 μM caffeine decreased total cell reactive oxygen species (ROS) production but increased mitochondrial ROS production. In contrast, caffeine (10 nM and 10 μM) protected cells against gemcitabine-induced toxicity and apoptosis. Similarly, 1 μM and 10 μM caffeine protected cells against paclitaxel-induced toxicity and mitochondrial ROS production. Chlorogenic acid had no effect on chemotherapy-induced toxicity in SH-SY5Y cells. In conclusion, this study provides preliminary evidence that caffeine, not chlorogenic acid, modulates the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in SH-SY5Y cells via different mechanisms.

  16. Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid.

    PubMed

    Silva, Alexandra; Sampaio-Marques, Belém; Fernandes, Angela; Carreto, Laura; Rodrigues, Fernando; Holcik, Martin; Santos, Manuel A S; Ludovico, Paula

    2013-01-01

    Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.

  17. Protective Effects and Mechanisms of Salvianolic Acid B Against H₂O₂-Induced Injury in Induced Pluripotent Stem Cell-Derived Neural Stem Cells.

    PubMed

    Shu, Tao; Pang, Mao; Rong, Limin; Liu, Chang; Wang, Juan; Zhou, Wei; Wang, Xuan; Liu, Bin

    2015-06-01

    Induced pluripotent stem cells (iPSCs) have the potential to differentiate into neural lineages. Salvianolic acid B (Sal B) is a commonly used, traditional Chinese medicine for enhancing neuroprotective effects, and has antioxidant, anti-inflammatory, and antiapoptotic properties. Here, we explore the potential mechanism of Sal B in protecting iPSC-derived neural stem cells (NSCs) against H2O2-induced injury. iPSCs were induced into NSCs, iPSC-derived NSCs were treated with 50 μM Sal B for 24.5 h and 500 μM H2O2 for 24 h. The resulting effects were examined by flow cytometry analysis, quantitative reverse-transcription polymerase chain reaction, and western blotting. Upon H2O2 exposure, Sal B significantly promoted cell viability and stabilization of the mitochondrial membrane potential. Sal B also visibly decreased the cell apoptotic ratio. In addition, Sal B markedly reduced expression of matrix metalloproteinase (MMP)-2 and -9, and phosphospecific signal transducer and activator of transcription 3 (p-STAT3), and increased the level of tissue inhibitor of metalloproteinase (TIMP)-2 in iPSC-derived NSCs induced by H2O2. These results suggest that Sal B protects iPSC-derived NSCs against H2O2-induced oxidative stress. The mechanisms of this stress tolerance may be attributed to modulation of the MMP/TIMP system and inhibition of the STAT3 signaling pathway.

  18. Modulatory Effect of Betulinic Acid on the Genotoxicity Induced by Different Mutagens in V79 Cells

    PubMed Central

    Acésio, Nathália Oliveira; de Oliveira, Pollyanna Francielli; Mastrocola, Daiane Fernanda Pereira; Lima, Ildercílio Mota de Souza; Munari, Carla Carolina; Sato, Vânia Luiza Ferreira Lucatti; Souza, Andressa Aparecida Silva; Flauzino, Lúzio Gabriel Bocalon; Cunha, Wilson Roberto; Tavares, Denise Crispim

    2016-01-01

    Betulinic acid (BA) is a pentacyclic triterpene that can be isolated from many medicinal plants around the world. The aim of this study was to evaluate the genotoxic potential of BA and its effect on the genotoxicity induced by different mutagens in V79 cells using the cytokinesis-block micronucleus assay. Different BA concentrations were combined with methyl methanesulfonate (MMS), doxorubicin (DXR), camptothecin (CPT), and etoposide (VP-16). The frequencies of micronuclei in cultures treated with different BA concentrations did not differ from those of the negative control. Treatment with BA and MMS resulted in lower micronucleus frequencies than those observed for cultures treated with MMS alone. On the other hand, a significant increase in micronucleus frequencies was observed in cultures treated with BA combined with DXR or VP-16 when compared to these mutagens alone. The results showed no effect of BA on CPT-induced genotoxicity. Therefore, BA was not genotoxic under the present experimental conditions and exerted a different influence on the genotoxicity induced by different mutagens. The modulatory effect of BA depends on the type of mutagen and concentrations used. PMID:27195016

  19. Salvianolic acid B, a novel autophagy inducer, exerts antitumor activity as a single agent in colorectal cancer cells

    PubMed Central

    Zhang, Lumin; Chen, Liuxi; Zhang, Xiaomin; Liang, Xiao; Xie, Jiansheng; Fang, Yong; Sui, Xinbing; Han, Weidong; Pan, Hongming

    2016-01-01

    Salvianolic Acid B (Sal B), an active compound extracted from the Chinese herb Salvia miltiorrhiza, is attracting more and more attention due to its biological activities, including antioxidant, anticoagulant and antitumor effects. However, autophagy induction in cancer cells by Sal B has never been recognized. In this study, we demonstrated that Sal B induced cell death and triggered autophagy in HCT116 and HT29 cells in a dose-dependent manner. Specific inhibition of autophagy by 3-MA or shRNA targeting Atg5 rescued Sal B-induced cell death in vitro and in vivo, suggesting that Sal B-induced autophagy may play a pro-death role and contribute to the cell death of colorectal cancer cell lines. Furthermore, AKT/mTOR signaling pathway was demonstrated to be a critical mediator in regulating Sal B-induced cell death. Overexpression of AKT by the transfection with AKT plasmid or pretreatment with insulin decreased Sal B-induced autophagy and cell death. Inversely, inhibition of AKT by LY294002 treatment markedly enhanced Sal B-induced autophagy and cell death. Taken together, our results demonstrate, for the first time, that Sal B is a novel autophagy inducer and exerts its antitumor activity as a single agent in colorectal cancer cells through the suppression of AKT/mTOR pathway. PMID:27557491

  20. Salvianolic acid B, a novel autophagy inducer, exerts antitumor activity as a single agent in colorectal cancer cells.

    PubMed

    Jing, Zhao; Fei, Weiqiang; Zhou, Jichun; Zhang, Lumin; Chen, Liuxi; Zhang, Xiaomin; Liang, Xiao; Xie, Jiansheng; Fang, Yong; Sui, Xinbing; Han, Weidong; Pan, Hongming

    2016-09-20

    Salvianolic Acid B (Sal B), an active compound extracted from the Chinese herb Salvia miltiorrhiza, is attracting more and more attention due to its biological activities, including antioxidant, anticoagulant and antitumor effects. However, autophagy induction in cancer cells by Sal B has never been recognized. In this study, we demonstrated that Sal B induced cell death and triggered autophagy in HCT116 and HT29 cells in a dose-dependent manner. Specific inhibition of autophagy by 3-MA or shRNA targeting Atg5 rescued Sal B-induced cell death in vitro and in vivo, suggesting that Sal B-induced autophagy may play a pro-death role and contribute to the cell death of colorectal cancer cell lines. Furthermore, AKT/mTOR signaling pathway was demonstrated to be a critical mediator in regulating Sal B-induced cell death. Overexpression of AKT by the transfection with AKT plasmid or pretreatment with insulin decreased Sal B-induced autophagy and cell death. Inversely, inhibition of AKT by LY294002 treatment markedly enhanced Sal B-induced autophagy and cell death. Taken together, our results demonstrate, for the first time, that Sal B is a novel autophagy inducer and exerts its antitumor activity as a single agent in colorectal cancer cells through the suppression of AKT/mTOR pathway.

  1. Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice

    SciTech Connect

    Okano, Junko . E-mail: okajun@anat1.med.kyoto-u.ac.jp; Suzuki, Shigehiko; Shiota, Kohei

    2007-05-15

    Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G{sub 1}/S progression of palatal mesenchymal cells through upregulation of p21 {sup Cip1}, leading to Rb hypophospholylation. Thus, RA appears to cause G{sub 1} arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.

  2. Hypoxia and retinoic acid-inducible NDRG1 expression is responsible for doxorubicin and retinoic acid resistance in hepatocellular carcinoma cells.

    PubMed

    Jung, Eun Uk; Yoon, Jung-Hwan; Lee, Youn-Jae; Lee, Jeong-Hoon; Kim, Bo Hyun; Yu, Su Jong; Myung, Sun Jung; Kim, Yoon Jun; Lee, Hyo-Suk

    2010-12-01

    Hypoxia may activate survival signals in cancer cells. Moreover, hypoxic cells are less sensitive than normoxic cells to doxorubicin cytotoxicity, a potent activator of the p53 tumor suppressor gene. N-myc downstream-regulated gene-1 (NDRG1) is a hypoxia- and retinoic acid-inducible protein, and has been previously implicated in carcinogenesis. As this protein is also a downstream target of p53 and hepatocellular carcinoma (HCC) cells frequently evidence resistance to retinoic acid (RA) cytotoxicity, we attempted to determine whether the suppression of NDRG1 expression may sensitize HCC cells to doxorubicin and/or RA cytotoxicity. HCC cells expressed NDRG1 protein, and the expression of this protein was hypoxia- and RA-inducible. Doxorubicin treatment induced HCC cell cytotoxicity via the activation of mitochondrial apoptotic signals, including caspase-9 activation. Hypoxic HCC cells are less sensitive to doxorubicin-induced apoptosis. The suppression of NDRG1 expression either by siRNA or flavopiridol sensitized hypoxic HCC cells to doxorubicin cytotoxicity, and this was attributed to more profound augmentation of JNK and caspase-9 activation. The suppression of NDRG1 expression also sensitized RA-resistant HCC cells to RA-induced apoptosis, and this sensitization was more apparent in hypoxic HCC cells than in normoxic cells. Glutaredoxin2 expression was down-regulated in NDRG1-suppressed HCC cells. These results show that hypoxia- and RA-inducible NDRG1 expression is responsible for doxorubicin and RA resistance in HCC cells. Thus, the selective interruption of NDRG1 signaling may prove to be therapeutically useful in HCCs, particularly in the advanced infiltrative type of tumors exposed to hypoxic environments.

  3. Maturation of murine bone marrow dendritic cells induced by acidic Ginseng polysaccharides.

    PubMed

    Wang, Zuozhou; Meng, Jingjuan; Xia, Yanjie; Meng, Yiming; Du, Lin; Zhang, Zhenjie; Wang, Enhua; Shan, Fengping

    2013-02-01

    In this study, we report that a acidic polysaccharide (AGP) isolated from a Chinese medicinal herb, named Ginseng (Panax giseng C.A. Meyer), induces maturation of bone marrow dendritic cells (BMDCs) via concrete changes both inside and outside BMDCs. The impacts of AGP on BMDCs were assessed with use of conventional scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) for morphology, flow cytometry (FCM) for key surface molecules, cytochemistry assay, FITC-dextran, bio-assay for phagocytosis and enzyme linked immunosorbent assay (ELISA) for production of cytokines. Our results elucidated that PPS promoted maturation of BMDCs via changes as reflected by the down-regulation of acid phosphatase (ACP) activity inside the BMDCs, which occurs when phagocytosis of BMDCs to antigen decreased, while antigen presentation increased upon maturation, higher expression of key surface molecules of MHC II, CD80, CD86, CD83, and CD40, and releasing higher level of cytokines IL-12 and low level of TNF-α. Our study suggest that AGP play marked immunostimulating role on the maturation of murine BMDCs through precise regulation of phagocytosis and enzyme activities inside the BMDCs.

  4. Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells.

    PubMed

    George, Kerri L; Saltman, Laura H; Stein, Gary S; Lian, Jane B; Zurier, Robert B

    2008-03-01

    Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.

  5. Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) contributes to retinoic acid-induced differentiation of leukemic cells

    SciTech Connect

    Yu, Yun; Shen, Shao-Ming; Zhang, Fei-Fei; Wu, Zhao-Xia; Han, Bin; Wang, Li-Shun

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer ANP32B was down-regulated during ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer Knockdown of ANP32B enhanced ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer Ectopic expression of ANP32B inhibited ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer ANP32B inhibited ATRA activated transcriptional activity of RAR{alpha}. -- Abstract: The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is a member of a conserved superfamily of nuclear proteins whose functions are largely unknown. In our previous work, ANP32B was identified as a novel direct substrate for caspase-3 and acted as a negative regulator for leukemic cell apoptosis. In this work, we provided the first demonstration that ANP32B expression was down-regulated during differentiation induction of leukemic cells by all-trans retinoic acid (ATRA). Knockdown of ANP32B expression by specific shRNA enhanced ATRA-induced leukemic cell differentiation, while ectopic expression of ANP32B attenuated it, indicating an inhibitory role of ANP32B against leukemic cell differentiation. Furthermore, luciferase reporter assay revealed that ANP32B might exert this role through inhibiting the ATRA dependent transcriptional activity of retinoic acid receptor (RAR{alpha}). These data will shed new insights into understanding the biological functions of ANP32B protein.

  6. Salicylic acid induces apoptosis in colon carcinoma cells grown in-vitro: Influence of oxygen and salicylic acid concentration

    SciTech Connect

    Zitta, Karina; Meybohm, Patrick; Bein, Berthold; Huang, Ying; Heinrich, Christin; Scholz, Jens; Steinfath, Markus; Albrecht, Martin

    2012-04-15

    In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10 {mu}M SA on CaCo-2 cells grown under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen. -- Highlights: Black-Right-Pointing-Pointer Effects of salicylic acid on colon carcinoma cells grown under normoxic and hypoxic conditions Black-Right-Pointing-Pointer Salicylic acid increases caspase-3/7 activity and hydrogen peroxide release under normoxia Black-Right-Pointing-Pointer Salicylic acid decreases pro-survival erk-1/2 phosphorylation under normoxia Black-Right-Pointing-Pointer Salicylic acid does

  7. Retinoic acid can induce mouse embryonic stem cell R1/E to differentiate toward female germ cells while oleanolic acid can induce R1/E to differentiate toward both types of germ cells.

    PubMed

    Wan, Qian; Lu, Hua; Wu, Lin-Tao; Liu, Xia; Xiang, Jun-Bei

    2014-12-01

    Retinoic acid (RA) and oleanolic acid (OA) were studied about their potential to induce mouse embryonic stem cell R1/E (MESC-R1/E) to differentiate toward germ cells. Embryoid bodies (EBs) first formed from MESC-R1/E and EBs were allowed to attach to the bottoms of normal cell-culturing plate and grow. Then, different compounds including RA, OA and so on were respectively added to induce MESC-R1/E to differentiate. After 72 h, microscopy images were taken for all interventions, then total RNAs were extracted, cDNAs were synthesized and real-time fluorescence quantitative PCR (qPCR) was performed to detect the transcriptional expression patterns of 11 reproductive-differentiation-related genes for different compounds respectively. During the data analysis, it was found RA significantly up-regulated the expression levels of GDF-9, Stra8, SCP3, Mvh, ZP1, ZP2, and ZP3, while significantly down-regulated the levels of Itag6 and Itgb1, and the level of Oct-4 was down-regulated insignificantly, while the level of TP2 was up-regulated insignificantly; OA significantly up-regulated the expression levels of Stra8, SCP3, Mvh, ZP1, ZP2, Itgb1, and TP2, and the levels of Oct-4, GDF-9, ZP3, and Itga6 were up-regulated insignificantly. The data showed that RA can induce MESC-R1/E to differentiate toward female germ cells while OA can induce MESC-R1/E to differentiate toward male and female germ cells.

  8. Statin induced myotoxicity: the lactone forms are more potent than the acid forms in human skeletal muscle cells in vitro.

    PubMed

    Skottheim, Ine Blankenberg; Gedde-Dahl, Ane; Hejazifar, Solmaz; Hoel, Kjersti; Asberg, Anders

    2008-04-23

    Statins exist in both acid and lactone forms in vivo. High plasma levels of the lactone forms have been observed in patients with statin induced myopathy. In the present study, the hypothesis that lactone forms have a higher potency of inducing myotoxicity as compared to acid forms was investigated. Primary human skeletal muscle cells were incubated with increasing concentrations of lactone and acid forms of atorvastatin, fluvastatin, pravastatin and simvastatin. Following incubation, living myotubes were quantified by fluorescence staining. Atorvastatin lactone showed a 14-fold, fluvastatin lactone a 26-fold, pravastatin lactone a 23-fold, and simvastatin lactone a 37-fold higher potency to induce myotoxicity compared to their corresponding acid forms. Thus, for the four different statins the present study shows a significantly higher potency of the lactone forms, than the respective acid forms, to induce myotoxicity in human skeletal muscle cells in vitro. These results clearly indicate the need to differentiate between acid and lactone forms in future investigation of statin myotoxicity.

  9. Apigenin reduce lipoteichoic acid-induced inflammatory response in rat cardiomyoblast cells.

    PubMed

    Gutiérrez-Venegas, Gloria; González-Rosas, Zeltzin

    2017-02-01

    Infective endocarditis is caused by Streptococcus sanguinis present in dental plaque, which can induce inflammatory responses in the endocardium. The present study depicts research on the properties of apigenin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from S. sanguinis. Interleukin-1β and cyclooxygenase (COX)-2 expression were detected by reverse transcriptase polymerase chain reaction. In addition, western blot assays and immuno-fluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, as well as activity of the mitogen activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Effect of apigenin on cell viability was equally assessed in other experimental series. Our results showed that apigenin blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA in a dose-dependent fashion. Moreover, apigenin showed no cytotoxic effects; it blocked NF-κB translocation and IκB degradation. Our findings suggested that apigenin possessed potential value in the treatment of infectious endocarditis.

  10. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    PubMed

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  11. Protective Effect of Unsaturated Fatty Acids on Palmitic Acid-Induced Toxicity in Skeletal Muscle Cells is not Mediated by PPARδ Activation.

    PubMed

    Tumova, Jana; Malisova, Lucia; Andel, Michal; Trnka, Jan

    2015-10-01

    Unsaturated free fatty acids (FFA) are able to prevent deleterious effects of saturated FFA in skeletal muscle cells although the mechanisms involved are still not completely understood. FFA act as endogenous ligands of peroxisome proliferator-activated receptors (PPAR), transcription factors regulating the expression of genes involved in lipid metabolism. The aim of this study was to determine whether activation of PPARδ, the most common PPAR subtype in skeletal muscle, plays a role in mediating the protective effect of unsaturated FFA on saturated FFA-induced damage in skeletal muscle cells and to examine an impact on mitochondrial respiration. Mouse C2C12 myotubes were treated for 24 h with different concentrations of saturated FFA (palmitic acid), unsaturated FFA (oleic, linoleic and α-linolenic acid), and their combinations. PPARδ agonist GW501516 and antagonist GSK0660 were also used. Both mono- and polyunsaturated FFA, but not GW501516, prevented palmitic acid-induced cell death. Mono- and polyunsaturated FFA proved to be effective activators of PPARδ compared to saturated palmitic acid; however, in combination with palmitic acid their effect on PPARδ activation was blocked and stayed at the levels observed for palmitic acid alone. Unsaturated FFA at moderate physiological concentrations as well as GW501516, but not palmitic acid, mildly uncoupled mitochondrial respiration. Our results indicate that although unsaturated FFA are effective activators of PPARδ, their protective effect on palmitic acid-induced toxicity is not mediated by PPARδ activation and subsequent induction of lipid regulatory genes in skeletal muscle cells. Other mechanisms, such as mitochondrial uncoupling, may underlie their effect.

  12. Oleic acid induces specific alterations in the morphology, gene expression and steroid hormone production of cultured bovine granulosa cells.

    PubMed

    Yenuganti, Vengala Rao; Viergutz, Torsten; Vanselow, Jens

    2016-06-01

    After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation.

  13. C/EBPβ: a major PML–RARA-responsive gene in retinoic acid-induced differentiation of APL cells

    PubMed Central

    Duprez, Estelle; Wagner, Katharina; Koch, Heike; Tenen, Daniel G.

    2003-01-01

    In acute promyelocytic leukemia (APL), the translocation t(15;17) induces a block at the promyelocytic stage of differentiation in an all-trans-retinoic acid (ATRA)-responsive manner. Here we report that upon treatment with ATRA, t(15;17) cells (NB4) reveal a very rapid increase in protein level and binding activity of C/EBPβ, a C/EBP family member, which was not observed in an ATRA-resistant NB4 cell line. We further provide evidence that ATRA mediates a direct increase of C/EBPβ, only in PML–RARA (promyelocytic leukemia–retinoic acid receptor α)-expressing cells. In addition, transactivation experiments indicate that the PML–RARA fusion protein, but not PML–RARA mutants defective in transactivation, strongly transactivates the C/EBPβ promoter. These results suggest that PML–RARA mediates ATRA-induced C/EBPβ expression. Finally, we demonstrate the importance of C/EBPβ in granulocytic differentiation. We show that not only does C/EBPβ induce granulocytic differentiation of non-APL myeloid cell lines independent of addition of ATRA or other cytokines, but also that C/EBPβ induction is required during ATRA-induced differentiation of APL cells. Taken together, C/EBPβ is an ATRA-dependent PML–RARA target gene involved in ATRA-induced differentiation of APL cells. PMID:14592978

  14. Impaired neural differentiation potency by retinoic acid receptor-α pathway defect in induced pluripotent stem cells.

    PubMed

    Hou, Pei-Shan; Huang, Wen-Chin; Chiang, Wei; Lin, Wei-Che; Chien, Chung-Liang

    2014-12-01

    Induced pluripotent stem cells (iPSCs) are reprogrammed from somatic cells via ectopic gene expression and, similarly to embryonic stem cells (ESCs), possess powerful abilities to self-renew and differentiate into cells of various lineages. However, the neural differentiation potency of iPSCs remains unknown. In this study, we demonstrated the neural differentiation ability of iPSCs compared with ESCs using an retinoic acid (RA) induction system. The neural differentiation efficiency of iPSCs was obviously lower than that of ESCs. Retinoic acid receptor-α (RARα) was critical in the RA-induced neural differentiation of iPSCs, and the effect of RARα was confirmed by applying a specific RARα antagonist ER50891 to ESCs. These findings indicate that iPSCs do not possess the complete properties that ESCs have.

  15. Gallic acid as a selective anticancer agent that induces apoptosis in SMMC-7721 human hepatocellular carcinoma cells.

    PubMed

    Sun, Guojun; Zhang, Shuqin; Xie, Yanru; Zhang, Ziyu; Zhao, Wenjing

    2016-01-01

    Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a naturally occurring plant polyphenol, isolated from water caltrop, which has been reported to exert anticancer effects. The present study investigated the antiproliferative effects of GA on the HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cell lines using MTT and colony formation assays. In particular, the underlying mechanism of GA-induced apoptosis in SMMC-7721 cells was studied in vitro by flow cytometry and western blotting. The results of the present study indicated that GA was capable of inhibiting the proliferation of HepG2 and SMMC-7721 cells in a time- and dose-dependent manner, as well as inducing the apoptosis of SMMC-7721 cells. GA induced caspase-3, caspase-9 and reactive oxygen species activity, elevated the expression of apoptosis regulator Bcl-2-like protein 4 and reduced the mitochondrial membrane potential in SMMC-7721 cells. When compared with HL-7702 normal human hepatocytes, GA demonstrated selective toxicity for HCC cells. In conclusion, GA is able to induce apoptosis in SMMC-7721 cells in vitro via mitochondrial-mediated pathways, and may possess the potential to be a novel therapeutic compound for use in the treatment of HCC.

  16. Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic β-Cells.

    PubMed

    Šrámek, Jan; Němcová-Fürstová, Vlasta; Kovář, Jan

    2016-09-12

    Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells.

  17. Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis.

    PubMed

    Englert, Robert P; Shacter, Emily

    2002-06-07

    Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms. In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl. The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated. When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2). Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2). In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis. HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg. The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.

  18. Acetic acid-induced programmed cell death and release of volatile organic compounds in Chlamydomonas reinhardtii.

    PubMed

    Zuo, Zhaojiang; Zhu, Yerong; Bai, Yanling; Wang, Yong

    2012-02-01

    Acetic acid widely spreads in atmosphere, aquatic ecosystems containing residues and anoxic soil. It can inhibit aquatic plant germination and growth, and even cause programmed cell death (PCD) of yeast. In the present study, biochemical and physiological responses of the model unicellular green algae Chlamydomonas reinhardtii were examined after acetic acid stress. H(2)O(2) burst was found in C. reinhardtii after acetic acid stress at pH 5.0 for 10 min. The photosynthetic pigments were degraded, gross photosynthesis and respiration were disappeared gradually, and DNA fragmentation was also detected. Those results indicated that C. reinhardtii cells underwent a PCD but not a necrotic, accidental cell death event. It was noticed that C. reinhardtii cells in PCD released abundant volatile organic compounds (VOCs) upon acetic acid stress. Therefore, we analyzed the VOCs and tested their effects on other normal cells. The treatment of C. reinhardtii cultures with VOCs reduced the cell density and increased antioxidant enzyme activity. Therefore, a function of VOCs as infochemicals involved in cell-to-cell communication at the conditions of applied stress is suggested.

  19. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    PubMed

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

  20. Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

    PubMed

    Romanov, Victor; Whyard, Terry C; Waltzer, Wayne C; Grollman, Arthur P; Rosenquist, Thomas

    2015-01-01

    Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells.

  1. Effect of mycophenolic acid on TNFα-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

    PubMed Central

    Hauser, Ingeborg A; Johnson, David R; Thévenod, Frank; Goppelt-Strübe, Margarete

    1997-01-01

    Mycophenolic acid (MPA) is an inhibitor of inosine-5′-monophosphate dehydrogenase and therefore interferes with cellular GTP biosynthesis. Recently, MPA has been used as an antiproliferative and immunosuppressive agent. In the present study, the effect of MPA on the expression of the endothelial cell adhesion molecules (CAMs), intercellular (I) CAM-1, vascular (V) CAM-1 and endothelial (E)-selectin, was investigated in tumour necrosis factor-α (TNFα)-activated cultured human venous endothelial cells (EC).Surface expression of CAMs was measured by flow cytometry and mRNA expression by Northern blot analysis. Transcriptional activation of CAMs by the nuclear factor NF-κB was determined by an electromobility shift assay. The function of CAMs was studied by a static adhesion assay with human monocyte-like undifferentiated U937 cells.Pretreatment of TNFα- (5  ng ml−1, 12 h) activated EC with MPA (10 μM, 24 h) increased the binding of U937 cells, which had not been treated with MPA, by ≈amp;2 fold. MPA-pretreatment of EC did not affect TNFα-induced surface expression of ICAM-1. However, VCAM-1 and E-selectin were increased 2–3 fold and remained elevated up to 24 h, by which time TNFα-activated control EC had returned to baseline levels of expression. The effect of MPA on the surface expression of CAMs was half-maximal at ≈amp;1 μM and required ⩾12 h of pretreatment. Guanosine (0.3 mM), a precursor of GTP, did not prevent the effect of MPA on the expression of CAMs in TNFα-activated EC.Kinetics of mRNA expression of CAMs mirrored protein expression: mRNA for ICAM-1 was unaffected, whereas TNFα-induced mRNA expression for E-selectin and VCAM-1 was prolonged and increased by MPA. This effect was not due to increased transcription mediated by the nuclear transcription factor NF-κB. However, half-life for E-selectin mRNA was increased 10 fold by MPA, whereas ICAM-1 mRNA half-life was unchanged.The data demonstrate that apart from its

  2. Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

    PubMed Central

    Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

    2011-01-01

    Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AX (γH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

  3. Deoxycholic acid causes DNA damage while inducing apoptotic resistance through NF-κB activation in benign Barrett's epithelial cells.

    PubMed

    Huo, Xiaofang; Juergens, Stefanie; Zhang, Xi; Rezaei, Davood; Yu, Chunhua; Strauch, Eric D; Wang, Jian-Ying; Cheng, Edaire; Meyer, Frank; Wang, David H; Zhang, Qiuyang; Spechler, Stuart J; Souza, Rhonda F

    2011-08-01

    Gastroesophageal reflux is associated with adenocarcinoma in Barrett's esophagus, but the incidence of this tumor is rising, despite widespread use of acid-suppressing medications. This suggests that refluxed material other than acid might contribute to carcinogenesis. We looked for potentially carcinogenetic effects of two bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on Barrett's epithelial cells in vitro and in vivo. We exposed Barrett's (BAR-T) cells to DCA or UDCA and studied the generation of reactive oxygen/nitrogen species (ROS/RNS); expression of phosphorylated H2AX (a marker of DNA damage), phosphorylated IkBα, and phosphorylated p65 (activated NF-κB pathway proteins); and apoptosis. During endoscopy in patients, we took biopsy specimens of Barrett's mucosa before and after esophageal perfusion with DCA or UDCA and assessed DNA damage and NF-κB activation. Exposure to DCA, but not UDCA, resulted in ROS/RNS production, DNA damage, and NF-κB activation but did not increase the rate of apoptosis in BAR-T cells. Pretreatment with N-acetyl-l-cysteine (a ROS scavenger) prevented DNA damage after DCA exposure, and DCA did induce apoptosis in cells treated with NF-κB inhibitors (BAY 11-7085 or AdIκB superrepressor). DNA damage and NF-κB activation were detected in biopsy specimens of Barrett's mucosa taken after esophageal perfusion with DCA, but not UDCA. These data show that, in Barrett's epithelial cells, DCA induces ROS/RNS production, which causes genotoxic injury, and simultaneously induces activation of the NF-κB pathway, which enables cells with DNA damage to resist apoptosis. We have demonstrated molecular mechanisms whereby bile reflux might contribute to carcinogenesis in Barrett's esophagus.

  4. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA).

    PubMed

    Gotoh, Mari; Sano-Maeda, Katsura; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2012-01-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2)) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl(2) induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2)-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2). Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1), LPA(2), and LPA(6), which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1) and LPA(2) have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1) and LPA(2) on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1) and LPA(3) antagonist, was adopted to know the LPA(1) function and siRNA was used to knockdown the expression of LPA(2). On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2)-induced hypoxia damage is mediated via LPA(2).

  5. Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway.

    PubMed

    Lu, Tien-Hui; Su, Chin-Chuan; Tang, Feng-Cheng; Chen, Chun-Hung; Yen, Cheng-Chieh; Fang, Kai-Min; Lee, kuan-I; Hung, Dong-Zong; Chen, Ya-Wen

    2015-01-05

    Chloroacetic acid (CA), a chlorinated analog of acetic acid and an environmental toxin that is more toxic than acetic, dichloroacetic, or trichloroacetic acids, is widely used in chemical industries. Furthermore, CA has been found to be the major disinfection by-products (DBPs) of drinking water. CA has been reported to be highly corrosive and to induce severe tissue injuries (including nervous system) that lead to death in mammals. However, the effects and underlying mechanisms of CA-induced neurotoxicity remain unknown. In the present study, we found that CA (0.5-2.0 mM) significantly increased LDH release, decreased the number of viable cells (cytotoxicity) and induced apoptotic events (including: increases in the numbers of apoptotic cells, the membrane externalization of phosphatidylserine (PS), and caspase-3/-7 activity) in Neuro-2a cells. CA (1.5 mM; the approximate to LD50) also triggered ER stress, which was identified by monitoring several key molecules that are involved in the unfolded protein responses (including the increase in the expressions of p-PERK, p-IRE-1, p-eIF2α, ATF-4, ATF-6, CHOP, XBP-1, GRP 78, GRP 94, and caspase-12) and calpain activity. Transfection of GRP 78- and GRP 94-specific si-RNA effectively abrogated CA-induced cytotoxicity, caspase-3/-7 and caspase-12 activity, and GRP 78 and GRP 94 expression in Neuro-2a cells. Additionally, pretreatment with 2.5 mM N-acetylcysteine (NAC; a glutathione (GSH) precursor) dramatically suppressed the increase in lipid peroxidation, cytotoxicity, apoptotic events, calpain and caspase-12 activity, and ER stress-related molecules in CA-exposed cells. Taken together, these results suggest that the higher concentration of CA exerts its cytotoxic effects in neuronal cells by triggering apoptosis via a ROS-induced ER stress signaling pathway.

  6. Suberoyl bishydroxamic acid-induced apoptosis in HeLa cells via ROS-independent, GSH-dependent manner.

    PubMed

    You, Bo Ra; Park, Woo Hyun

    2013-05-01

    Suberoyl bishydroxamic acid (SBHA) is a HDAC inhibitor that can regulate many biological functions including apoptosis and proliferation in various cancer cells. Here, we evaluated the effect of SBHA on the growth of HeLa cervical cancer cells in relation to apoptosis, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC50 of approximately 15 μM at 72 h. SBHA also induced apoptosis in HeLa cells, as evidenced by sub-G1 cells, annexin V-FITC staining cells, activations of caspase 3 and 8, and the loss of mitochondrial membrane potential (ΔΨm). In addition, all of the tested caspase inhibitors rescued some cells from SBHA-induced HeLa cell death. SBHA increased ROS levels including O2(•-) and induced GSH depletion in HeLa cells. Generally, caspase inhibitors did not affect ROS levels in SBHA-treated HeLa cells, but they significantly prevented GSH depletion in these cells. Furthermore, while the well-known antioxidants, N-acetyl cysteine and vitamin C, did not affect cell death, ROS level or GSH depletion in SBHA-treated HeLa cells, L-buthionine sulfoximine, a GSH synthesis inhibitor, enhanced cell death and GSH depletion in these cells. In conclusion, SBHA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis, and the inhibition is independent of ROS level changes, but dependent on GSH level changes.

  7. ERK inhibition sensitizes cancer cells to oleanolic acid-induced apoptosis through ERK/Nrf2/ROS pathway.

    PubMed

    Liu, Jia; Ma, Leina; Chen, Xiao; Wang, Jianxun; Yu, Tao; Gong, Ying; Ma, Aiguo; Zheng, Lanhong; Liang, Hui

    2016-06-01

    Oleanolic acid (OA) is a natural triterpenoid that is widely distributed in edible and medicinal plants. OA exerts anti-tumor activity on a wide range of cancer cells primarily through inducing apoptosis. Dysregulated ERK signaling is closely complicated in the biology of cancer, such as metastasis, proliferation, and survival, and it can be activated by various stimuli. In this study, we found that OA induced the activation of ERK in cancer cells. ERK activation compromised the apoptosis induced by OA. Blocking ERK activation by U0126 or siRNAs was able to potentiate the pro-apoptotic activity of OA on cancer cells. OA was shown to promote ERK-dependent Nrf2 expression in cancer cells, and in turn, Nrf2 expression was able to suppress OA-induced ROS generation. Blockade of Nrf2 expression was able to increase ROS levels and apoptotic death in cancer cells. In conclusion, we provided evidences that ERK activation is a mechanism underlying the resistance of cancer cells to OA-induced apoptosis and targeting ERK is a promising strategy to enhance the anti-tumor efficacy of OA.

  8. Hydrogen peroxide-induced apoptosis of HL-60 human leukemia cells is mediated by the oxidants hypochlorous acid and chloramines.

    PubMed

    Wagner, Brett A; Britigan, Bradley E; Reszka, Krzysztof J; McCormick, Michael L; Burns, C Patrick

    2002-05-15

    We set out to identify whether HOCl, which is generated from H(2)O(2) /MPO/Cl(-), is a proximal mediator of H(2)O(2) programmed cell death in the HL-60 human leukemia cell. We found that authentic HOCl induces apoptosis in the HL-60 cell. Both the addition of methionine, an HOCl scavenger, and the removal of Cl(-) from the medium to prevent the formation of HOCl inhibited H(2)O(2)-induced apoptosis. HL-60 cells underwent apoptosis when exposed to HOCl in full medium, which gives rise to chloramines by the reaction of HOCl with amine groups, but not by HOCl in the amine-free HBSS, in which HOCl but not chloramines can be detected. Authentic chloramines induced apoptosis in this cell line in a concentration-dependent manner and at concentrations lower than HOCl. Full medium exposed to HOCl for 24 h would support methionine noninhibitable apoptosis, but did not react with 2-nitro-5-thiobenzoic acid (TNB), raising the possibility that the final inducer is a nonoxidant formed from HOCl and chloramines. We conclude that the signal for apoptosis induced by H(2)O(2) in the MPO-containing HL-60 cell involves the reaction of the diffusible oxidant HOCl with amines producing chloramines and a subsequent non-TNB-reactive product.

  9. Perfluorooctanoic acid induces human Ishikawa endometrial cancer cell migration and invasion through activation of ERK/mTOR signaling

    PubMed Central

    Li, Fujun; Wang, Yixong; Xu, Yang; Zhang, Mei; Zhang, Xiaoqian; Ying, Xiaoyan; Zhang, Xuesen

    2016-01-01

    Perfluorooctanoic acid (PFOA) is a common environmental pollutant that has been associated with various diseases, including cancer. We explored the molecular mechanisms underlying PFOA-induced endometrial cancer cell invasion and migration. PFOA treatment enhanced migration and invasion by human Ishikawa endometrial cancer cells, which correlated with decreased E-cadherin expression, a marker of epithelial-mesenchymal transition. PFOA also induced activation of ERK1/2/mTOR signaling. Treatment with rapamycin, an mTOR inhibitor, antagonized the effects of PFOA and reversed the effects of PFOA activation in a xenograft mouse model of endometrial cancer. Consistent with these results, pre-treatment with rapamycin abolished PFOA-induced down-regulation of E-cadherin expression. These results indicate that PFOA is a carcinogen that promotes endometrial cancer cell migration and invasion through activation of ERK/mTOR signaling. PMID:27589685

  10. Zoledronic acid exerts antitumor effects in NB4 acute promyelocytic leukemia cells by inducing apoptosis and S phase arrest.

    PubMed

    Liu, Shou-Sheng; Wang, Xiao-Pai; Li, Xiu-Bo; Liang, Jia-Yi; Liu, Ling-Ling; Lu, Ying; Zhong, Xue-Yun; Chen, Yun-Xian

    2014-10-01

    The aim of this study was to investigate the antitumor effect of zoledronic acid (ZOL) in the NB4 human acute promyelocytic leukemia (APL) cell line and explore the potential mechanism of action of this compound. NB4 cells were exposed to various concentrations (0-200μM) of ZOL. Cell viability was measured by MTS assay. The extent of cell apoptosis and distribution of cells in the different phases of the cell cycle were analyzed with flow cytometry. The expression of apoptosis- and cell cycle-related proteins was assayed by Western blot. The combined effect of ZOL and arsenic trioxide (ATO) on the proliferation of NB4 cells was also determined. The results of this study indicate that ZOL inhibits cell proliferation in a time- and dose-dependent fashion and also induces apoptosis and S phase arrest in a dose-dependent manner. The Western blot analysis confirmed the induction of apoptosis and S phase arrest, revealing that the pro-apoptosis proteins Bax, Puma and activated caspase-9 were upregulated and the anti-apoptosis proteins Bcl-2 and Bcl-xL were downregulated. ZOL at a concentration of 50μM synergized with 0.5μM ATO on the growth inhibition of NB4 cells. Taken together, our data indicate that ZOL exerts a potent antitumor effect on NB4 cells by inducing apoptosis and cell cycle arrest, and that ZOL can synergize with the traditional chemotherapy drug ATO.

  11. Chronically elevated levels of short-chain fatty acids induce T cell-mediated ureteritis and hydronephrosis1

    PubMed Central

    Park, Jeongho; Goergen, Craig J.; HogenEsch, Harm; Kim, Chang H.

    2016-01-01

    Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10+ regulatory T cells, which may promote immune tolerance. However, SCFAs can also induce Th1 and Th17 cells upon immunological challenges and, therefore, also have the potential to induce inflammatory responses. Because of the seemingly paradoxical SCFA activities in regulating T cells, we investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and induced T-cell mediated ureteritis leading to kidney hydronephrosis (hereafter called C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction (UPJ) and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mTOR activation, T cell-derived inflammatory cytokines such as IFNγ and IL-17, and gut microbiota. Young or male animals were more susceptible than old or female animals respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Thus, SCFAs, when systemically administered at levels higher than physiological levels, cause dysregulated T cell responses and tissue inflammation in the renal system. The results provide insights into the immunological and pathological effects of chronically elevated SCFAs. PMID:26819206

  12. Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

    PubMed

    Rossato, Franco A; Zecchin, Karina G; La Guardia, Paolo G; Ortega, Rose M; Alberici, Luciane C; Costa, Rute A P; Catharino, Rodrigo R; Graner, Edgard; Castilho, Roger F; Vercesi, Aníbal E

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce

  13. Fatty Acid Synthase Inhibitors Induce Apoptosis in Non-Tumorigenic Melan-A Cells Associated with Inhibition of Mitochondrial Respiration

    PubMed Central

    Rossato, Franco A.; Zecchin, Karina G.; La Guardia, Paolo G.; Ortega, Rose M.; Alberici, Luciane C.; Costa, Rute A. P.; Catharino, Rodrigo R.; Graner, Edgard; Castilho, Roger F.; Vercesi, Aníbal E.

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat

  14. Eosinophils from Murine Lamina Propria Induce Differentiation of Naïve T Cells into Regulatory T Cells via TGF-β1 and Retinoic Acid.

    PubMed

    Chen, Hong-Hu; Sun, Ai-Hua; Ojcius, David M; Hu, Wei-Lin; Ge, Yu-Mei; Lin, Xu'ai; Li, Lan-Juan; Pan, Jian-Ping; Yan, Jie

    2015-01-01

    Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP) that displayed high aldehyde dehydrogenase (ALDH) activity, a rate-limiting step during all-trans retinoic acid (ATRA) synthesis, and expressed TGF-β1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-β1 or ATRA. Peripheral blood (PB) eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-β1 and ATRA.

  15. Palmitic acid induces osteoblastic differentiation in vascular smooth muscle cells through ACSL3 and NF-κB, novel targets of eicosapentaenoic acid.

    PubMed

    Kageyama, Aiko; Matsui, Hiroki; Ohta, Masahiko; Sambuichi, Keisuke; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Yokoyama, Tomoyuki; Kurabayashi, Masahiko

    2013-01-01

    Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited

  16. Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

    PubMed Central

    Tai, Ta-Wei; Chen, Ching-Yu; Su, Fong-Chin; Tu, Yuan-Kun; Tsai, Tsung-Ting; Lin, Chiou-Feng; Jou, I.-Ming

    2017-01-01

    Inhibiting osteoclasts and osteoclast precursors to reduce bone resorption is an important strategy to treat osteoclast-related diseases, such as osteoporosis, inflammatory bone loss, and malignant bone metastasis. However, the mechanism by which apoptosis is induced in the osteoclasts and their precursors are not completely understood. Here, we used nitrogen-containing bisphosphonate zoledronic acid (ZA) to induce cell apoptosis in human and murine osteoclast precursors and mature osteoclast-like cells. Caspase-3-mediated cell apoptosis occurred following the ZA (100 μM) treatment. Reactive oxygen species (ROS) were also generated in a time-dependent manner. Following knock-down of the p47phox expression, which is required for ROS activation, or co-treatment with the ROS inhibitor, N-acetyl-L-cysteine, ZA-induced apoptosis was significantly suppressed in both osteoclast precursors and mature osteoclast-like cells. The ROS-activated mitogen-activated protein kinases pathways did not trigger cell apoptosis. However, a ROS-regulated Mcl-1 decrease simultaneously with glycogen synthase kinase (GSK)-3β promoted cell apoptosis. These findings show that ZA induces apoptosis in osteoclast precursors and mature osteoclast-like cells by triggering ROS- and GSK-3β-mediated Mcl-1 down-regulation. PMID:28281643

  17. A Novel Tetraenoic Fatty Acid Isolated from Amaranthus spinosus Inhibits Proliferation and Induces Apoptosis of Human Liver Cancer Cells

    PubMed Central

    Mondal, Arijit; Guria, Tanmoy; Maity, Tapan Kumar; Bishayee, Anupam

    2016-01-01

    Amaranthus spinosus Linn. (Family: Amaranthaceae) has been shown to be useful in preventing and mitigating adverse pathophysiological conditions and complex diseases. However, only limited information is available on the anticancer potential of this plant. In this study, we examined the antiproliferative and pro-apoptotic effects of a novel fatty acid isolated from A. spinosus—(14E,18E,22E,26E)-methyl nonacosa-14,18,22,26 tetraenoate—against HepG2 human liver cancer cells. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine cell viability, flow cytometry assay for cell cycle analysis, and Western blot analysis to measure protein expression of Cdc2), cyclin B1, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2). The MTT assay showed that the fatty acid markedly inhibited the proliferation of HepG2 cells in a dosage-dependent fashion, with a half maximal inhibitory concentration (IC50) value of 25.52 µmol/L. This antiproliferative result was superior to that of another known fatty acid, linoleic acid (IC50 38.65 µmol/L), but comparable to that of standard anticancer drug doxorubicin (IC50 24.68 µmol/L). The novel fatty acid also induced apoptosis mediated by downregulation of cyclin B1, upregulation of Bax, and downregulation of Bcl-2, resulting in the G2/M transition arrest. Our results provide the first experimental evidence that a novel fatty acid isolated from A. spinosus exhibits significant antiproliferative activity mediated through the induction of apoptosis in HepG2 cells. These encouraging results may facilitate the development of A. spinosus fatty acid for the prevention and intervention of hepatocellular carcinoma. PMID:27669220

  18. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS-Ca(2+)-JNK mitochondrial pathways.

    PubMed

    Zhang, Yuanyuan; Han, Lirong; Qi, Wentao; Cheng, Dai; Ma, Xiaolei; Hou, Lihua; Cao, Xiaohong; Wang, Chunling

    2015-01-24

    Eicosapentaenoic acid (EPA), a well-known dietary n-3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca(2+)]c accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca(2+)]c generation, moreover, generation of ROS, overload of mitochondrial [Ca(2+)]c, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS-Ca(2+)-JNK mitochondrial pathways.

  19. Interaction between caspase-8 activation and endoplasmic reticulum stress in glycochenodeoxycholic acid-induced apoptotic HepG2 cells.

    PubMed

    Iizaka, Toru; Tsuji, Mayumi; Oyamada, Hideto; Morio, Yuri; Oguchi, Katsuji

    2007-11-30

    The accumulation of hydrophobic bile acid, such as glycochenodeoxycholic acid (GCDCA), in the liver has been thought to induce hepatocellular damage in human chronic cholestatic liver diseases. We previously reported that GCDCA-induced apoptosis was promoted by both mitochondria-mediated and endoplasmic reticulum (ER) stress-associated pathways in rat hepatocytes. In this study, we elucidated the relationship between these pathways in GCDCA-induced apoptotic HepG2 cells. HepG2 cells were treated with GCDCA (100-500microM) with or without a caspase-8 inhibitor, Z-IETD-fluoromethyl ketone (Z-IETD-FMK) (30microM) for 3-24h. We demonstrated the presence of both apoptotic pathways in these cells; that is, we showed increases in cleaved caspase-3 proteins, the release of cytochrome c from mitochondria, and the expression of ER resident molecular chaperone Bip mRNA and ER stress response-associated transcription factor Chop mRNA. On the other hand, pretreatment with Z-IETD-FMK significantly reduced the increases, compared with treatment with GCDCA alone. Immunofluorescence microscopic analysis showed that treatment with GCDCA increased the cleavage of BAP31, an integral membrane protein of ER, and pretreatment with Z-IETD-FMK suppressed the increase of caspase-8 and BAP31 cleavage. In conclusion, these results suggest that intact activated caspase-8 may promote and amplify the ER stress response by cleaving BAP31 in GCDCA-induced apoptotic cells.

  20. Alpha Cyano-4-Hydroxy-3-Methoxycinnamic Acid Inhibits Proliferation and Induces Apoptosis in Human Breast Cancer Cells

    PubMed Central

    Hamdan, Lamia; Arrar, Zoheir; Al Muataz, Yacoub; Suleiman, Lutfi; Négrier, Claude; Mulengi, Joseph Kajima; Boukerche, Habib

    2013-01-01

    This study investigated the underlying mechanism of 4-hydroxy-3-methoxycinnamic acid (ACCA), on the growth of breast cancer cells and normal immortal epithelial cells, and compared their cytotoxic effects responses. Treatment of breast cancer cells (MCF-7, T47D, and MDA-231) with ACCA resulted in dose- and time-dependent decrease of cell proliferation, viability in colony formation assay, and programmed cell death (apoptosis) with minimal effects on non-tumoral cells. The ability of ACCA to suppress growth in cancer cells not expressing or containing defects in p53 gene indicates a lack of involvement of this critical tumor suppressor element in mediating ACCA-induced growth inhibition. Induction of apoptosis correlated with an increase in Bax protein, an established inducer of programmed cell death, and the ratio of Bax to Bcl-2, an established inhibitor of apoptosis. We also documented the ability of ACCA to inhibit the migration and invasion of MDA-231 cells with ACCA in vitro. Additionally, tumor growth of MDA-231 breast cancer cells in vivo was dramatically affected with ACCA. On the basis of its selective anticancer inhibitory activity on tumor cells, ACCA may represent a promising therapeutic drug that should be further evaluated as a chemotherapeutic agent for human breast cancer. PMID:24039831

  1. Alpha cyano-4-hydroxy-3-methoxycinnamic acid inhibits proliferation and induces apoptosis in human breast cancer cells.

    PubMed

    Hamdan, Lamia; Arrar, Zoheir; Al Muataz, Yacoub; Suleiman, Lutfi; Négrier, Claude; Mulengi, Joseph Kajima; Boukerche, Habib

    2013-01-01

    This study investigated the underlying mechanism of 4-hydroxy-3-methoxycinnamic acid (ACCA), on the growth of breast cancer cells and normal immortal epithelial cells, and compared their cytotoxic effects responses. Treatment of breast cancer cells (MCF-7, T47D, and MDA-231) with ACCA resulted in dose- and time-dependent decrease of cell proliferation, viability in colony formation assay, and programmed cell death (apoptosis) with minimal effects on non-tumoral cells. The ability of ACCA to suppress growth in cancer cells not expressing or containing defects in p53 gene indicates a lack of involvement of this critical tumor suppressor element in mediating ACCA-induced growth inhibition. Induction of apoptosis correlated with an increase in Bax protein, an established inducer of programmed cell death, and the ratio of Bax to Bcl-2, an established inhibitor of apoptosis. We also documented the ability of ACCA to inhibit the migration and invasion of MDA-231 cells with ACCA in vitro. Additionally, tumor growth of MDA-231 breast cancer cells in vivo was dramatically affected with ACCA. On the basis of its selective anticancer inhibitory activity on tumor cells, ACCA may represent a promising therapeutic drug that should be further evaluated as a chemotherapeutic agent for human breast cancer.

  2. Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

    SciTech Connect

    Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien; Ju, Tsai-Kai; Huang, Yuan-Li; Lee, Ming-Shyue; Chen, Jiun-Hong; Lee, Hsinyu

    2013-11-01

    Highlights: •LPA induces ROS generation through LPA{sub 1} and LPA{sub 3}. •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA{sub 1} and LPA{sub 3} siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.

  3. Omega-3 fatty acids, EPA and DHA induce apoptosis and enhance drug sensitivity in multiple myeloma cells but not in normal peripheral mononuclear cells.

    PubMed

    Abdi, J; Garssen, J; Faber, J; Redegeld, F A

    2014-12-01

    The n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to enhance the effect of chemotherapeutic drugs in clinical studies in cancer patients and to induce apoptotic tumor cell death in vitro. Until now, EPA and DHA have never been investigated in multiple myeloma (MM). Human myeloma cells (L363, OPM-1, OPM-2 and U266) and normal peripheral blood mononuclear cells were exposed to EPA and DHA, and effects on mitochondrial function and apoptosis, caspase-3 activation, gene expression and drug toxicity were measured. Exposure to EPA and DHA induced apoptosis and increased sensitivity to bortezomib in MM cells. Importantly, they did not affect viability of normal human peripheral mononuclear cells. Messenger RNA expression arrays showed that EPA and DHA modulated genes involved in multiple signaling pathways including nuclear factor (NF) κB, Notch, Hedgehog, oxidative stress and Wnt. EPA and DHA inhibited NFκB activity and induced apoptosis through mitochondrial perturbation and caspase-3 activation. Our study suggests that EPA and DHA induce selective cytotoxic effects in MM and increase sensitivity to bortezomib and calls for further exploration into a potential application of these n-3 polyunsaturated fatty acids in the therapy of MM.

  4. The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

    PubMed

    Valenti, G; Procino, G; Carmosino, M; Frigeri, A; Mannucci, R; Nicoletti, I; Svelto, M

    2000-06-01

    Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

  5. Lactic and hydrochloric acids induce different patterns of inflammatory response in LPS-stimulated RAW 264.7 cells.

    PubMed

    Kellum, John A; Song, Mingchen; Li, Jinyou

    2004-04-01

    Metabolic acidosis frequently complicates sepsis and septic shock and may be deleterious to cellular function. Different types of metabolic acidosis (e.g., hyperchloremic and lactic acidosis) have been associated with different effects on the immune response, but direct comparative studies are lacking. Murine macrophage-like RAW 264.7 cells were cultured in complete medium with lactic acid or HCl to adjust the pH between 6.5 and 7.4 and then stimulated with LPS (Escherichia coli 0111:B4; 10 ng/ml). Nitric oxide (NO), IL-6, and IL-10 levels were measured in the supernatants. RNA was extracted from the cell pellets, and RT-PCR was performed to amplify corresponding mediators. Gel shift assay was also performed to assess NF-kappa B DNA binding. Inc easing concentrations of acid caused increasing acidification of the media. Trypan blue exclusion and lactate dehydrogenase release demonstrated that acidification did not reduce cell viability. HCl significantly increased LPS-induced NO release and NF-kappa B DNA binding at pH 7.0 but not at pH 6.5. IL-6 and IL-10 expression (RNA and protein) were reduced with HCl-induced acidification, but IL-10 was reduced much more than IL-6 at low pH. By contrast, lactic acid significantly decreased LPS-induced NO, IL-6, and IL-10 expression in a dose-dependent manner. Lactic acid also inhibited LPS-induced NF-kappa B DNA binding. Two common forms of metabolic acidosis (hyperchloremic and lactic acidosis) are associated with dramatically different patterns of immune response in LPS-stimulated RAW 264.7 cells. HCl is essentially proinflammatory as assessed by NO release, IL-6-to-IL-10 ratios, and NF-kappa B DNA binding. By contrast, lactic acidosis is anti-inflammatory.

  6. Suberoylanilide hydroxamic acid-induced HeLa cell death is closely correlated with oxidative stress and thioredoxin 1 levels.

    PubMed

    You, Bo Ra; Park, Woo Hyun

    2014-05-01

    Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor which has anticancer effects. We evaluated the growth inhibitory effects of SAHA on HeLa cervical cancer cells in relation to reactive oxygen species (ROS) levels. SAHA inhibited the growth of HeLa cells with an IC(50) of approximately 10 µM at 24 h, and induced apoptosis which was accompanied by the cleavage of PARP, caspase-3 activation and loss of mitochondrial membrane potential (MMP; ∆ψ(m)). All the tested caspase inhibitors prevented HeLa cell death induced by SAHA whereas TNF-α intensified apoptotic cell death in SAHA-treated HeLa cells. With respect to ROS and glutathione (GSH) levels, SAHA increased ROS levels, especially mitochondrial O(2)•- in HeLa cells and also induced GSH depletion. Caspase inhibitors reduced the levels of ROS and GSH depletion in SAHA-treated HeLa cells whereas TNF-α enhanced the levels in these cells. The well-known antioxidant N-acetyl cysteine (NAC) attenuated cell death and an increase in ROS levels was caused by SAHA. Moreover, SAHA decreased the levels of thioredoxin 1 (Trx1) in HeLa cells. While the downregulation of Trx1 enhanced cell death and ROS levels in SAHA-treated HeLa cells, the overexpression of Trx1 attenuated the levels in these cells. In conclusion, SAHA inhibited the growth of HeLa cell via caspase-dependent apoptosis, which was influenced by the mitochondrial O(2)•- and Trx1 levels.

  7. Relief of delayed oxidative stress by ascorbic acid can suppress radiation-induced cellular senescence in mammalian fibroblast cells.

    PubMed

    Kobashigawa, Shinko; Kashino, Genro; Mori, Hiromu; Watanabe, Masami

    2015-03-01

    Ionizing radiation-induced cellular senescence is thought to be caused by nuclear DNA damage that cannot be repaired. However, here we found that radiation induces delayed increase of intracellular oxidative stress after irradiation. We investigated whether the relief of delayed oxidative stress by ascorbic acid would suppress the radiation-induced cellular senescence in Syrian golden hamster embryo (SHE) cells. We observed that the level of oxidative stress was drastically increased soon after irradiation, then declined to the level in non-irradiated cells, and increased again with a peak on day 3 after irradiation. We found that the inductions of cellular senescence after X-irradiation were reduced along with suppression of the delayed induction of oxidative stress by treatment with ascorbic acid, but not when oxidative stress occurred immediately after irradiation. Moreover, treatment of ascorbic acid inhibited p53 accumulation at 3 days after irradiation. Our data suggested a delayed increase of intracellular oxidative stress levels plays an important role in the process of radiation-induced cellular senescence by p53 accumulation.

  8. Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1.

    PubMed

    Guaragnella, Nicoletta; Bobba, Antonella; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-01-04

    To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Deltayca1), cytochrome c (Deltacyc1,7) and both (Deltacyc1,7Deltayca1) were compared for AA-PCD occurrence, hydrogen peroxide (H(2)O(2)) production and caspase activity. AA-PCD occurs in Deltacyc1,7 and Deltacyc1,7Deltayca1 cells slower than in wt, but similar to that in Deltayca1 cells, in which no cytochrome c release occurs. Both H(2)O(2) production and caspase activation occur in these cells with early and extra-activation in Deltacyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.

  9. Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

    PubMed Central

    Li, Chen; Lun, Weijian; Zhao, Xinmei; Lei, Shan; Guo, Yandong; Ma, Jiayi

    2015-01-01

    Background. Allicin has anti-inflammatory, antioxidative and proapoptotic properties. Aims. To evaluate the effects and investigate the mechanism of allicin on trinitrobenzenesulfonic acid-induced colitis, specifically with mesalazine or sulfasalazine. Methods. 80 rats were divided equally into 8 groups: control; trinitrobenzenesulfonic acid; allicin prevention; allicin; mesalazine; sulfasalazine; allicin + sulfasalazine, and mesalazine + allicin. Systemic and colonic inflammation parameters were analysed. In addition, protein and culture medium of Caco-2 cells treated with various concentrations of IL-1β or allicin were collected for investigation of IL-8, NF-κB p65 P38, ERK, and JNK. One-way ANOVA and Kruskal-Wallis H test were used for parametric and nonparametric tests, respectively. Results. Allicin reduced the body weight loss of trinitrobenzenesulfonic acid-induced rats, histological score, serum TNF-α and IL-1β levels, and colon IL-1β mRNA level and induced serum IL-4 level, particularly in combination with mesalazine. In addition, 1 ng/mL IL-1β stimulated the P38, ERK, and JNK pathways, whereas pretreatment with allicin depressed this phenomenon, except for the ERK pathway. Conclusions. The inflammation induced by trinitrobenzenesulfonic acid is mitigated significantly by allicin treatment, particularly combined with mesalazine. Allicin inhibits the P38 and JNK pathways and the expression of NF-κB which explained the potential anti-inflammatory mechanisms of allicin. PMID:25729217

  10. Involvement of abscisic acid in ozone-induced puerarin production of Pueraria thomsnii Benth. suspension cell cultures.

    PubMed

    Sun, Lina; Su, Hu; Zhu, Yun; Xu, Maojun

    2012-01-01

    Exposure to ozone induced a rapid increase in the levels of the sesquiterpene phytohormone abscisic acid (ABA) and the isoflavone puerarin in suspension cell cultures of Pueraria thomsnii Benth. The observed increases in ABA and puerarin were dependent on the concentration of ozone applied to P. thomsnii cell cultures. In order to examine the role of ABA in ozone-induced puerarin production, cell suspensions were pretreated with the ABA biosynthetic inhibitor fluridone. Following ozone exposure, fluridone treatment suppressed ABA accumulation suggesting ABA was normally synthesized de novo through the carotenoid pathway. Fluridone also blocked ozone-induced puerarin production, which could be reversed through application of exogenous ABA. However, in the absence of ozone, ABA itself had no effect on puerarin accumulation in the suspension cells. Taken together, the data indicate that ozone is an efficient elicitor of puerarin production and may be particularly applicable for improving puerarin production in plant cell cultures. Furthermore, we demonstrate that ABA is one factor associated with ozone-induced puerarin production in P. thomsnii cell cultures.

  11. Omega-3 Polyunsaturated Fatty Acids Enhance Cisplatin Efficacy in Gastric Cancer Cells by Inducing Apoptosis via ADORA1.

    PubMed

    Sheng, Hong; Chen, Xuehua; Liu, Binya; Li, Pu; Cao, Weixin

    2016-01-01

    It has been suggested that administration of the omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can alter the toxicity and/or activity of several anticancer drugs in in vitro and in vivo studies. Here, we investigated the ability of ω-3 PUFAs to potentiate the antineoplastic activity of cisplatin (CDDP) in gastric cancer cells. The increase in CDDP-induced growth inhibition was measured by the IC50 values obtained when the cells were incubated with CDDP alone or with CDDP plus DHA or EPA. DHA and EPA enhanced the growth-inhibition activity of increasing concentrations of CDDP. The interactions between CDDP and DHA or EPA at the cellular level were assessed through the combination index (CI) method of Chou-Talalay. The results demonstrated synergism between CDDP and DHA or EPA in MKN45 cells. Cell cycle analysis showed that the combination treatment increased G0/G1 phase and S phase arrest, and significantly increased the number of apoptotic cells. According to our previous study, ω -3 PUFAs induce apoptosis of gastric cells via ADORA1, a subtype of adenosine receptor functionally related to cell death. The ADORA1 mRNA and protein expression was higher in the combination treatment than in the individual treatments. Notable, when GC cells were pretreated with DPCPX, a selective ADORA1 antagonist, the combination treatment effect on apoptosis was significantly reduced. Our results suggest that ω-3 PUFAs enhance the antineoplastic effects of CDDP in gastric cancer cells, and the synergistic effect between ω-3 PUFAs and CDDP is partly dependent on activating the ADORA1-mediated apoptosis pathway.

  12. A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

    PubMed

    Hirako, Naomi; Nakano, Hiroko; Takahashi, Shinichiro

    2014-01-01

    We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

  13. Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells.

    PubMed

    Xu, Q; Chen, S Y; Deng, L D; Feng, L P; Huang, L Z; Yu, R R

    2013-11-18

    Excessive oxidative stress in pancreatic β cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides.

  14. Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells.

    PubMed

    Yang, Jian; Wang, Wei; Ooi, Jolene; Campos, Lia S; Lu, Liming; Liu, Pentao

    2015-05-01

    We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog-1 (LRH1 or NR5A2) with OCT4, MYC, KLF4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration-free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose-sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant-negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell-like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β-catenin. These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency. Stem Cells 2015;33:1390-1404.

  15. Ammodytoxins efficiently release arachidonic acid and induce apoptosis in a motoneuronal cell line in an enzymatic activity-dependent manner.

    PubMed

    Jenko-Pražnikar, Zala; Petan, Toni; Pungerčar, Jože

    2013-03-01

    Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

  16. Beyond gastric acid reduction: Proton pump inhibitors induce heme oxygenase-1 in gastric and endothelial cells

    SciTech Connect

    Becker, Jan C. . E-mail: beckeja@uni-muenster.de; Grosser, Nina; Waltke, Christian; Schulz, Stephanie; Erdmann, Kati; Domschke, Wolfram; Schroeder, Henning; Pohle, Thorsten

    2006-07-07

    Proton pump inhibitors (PPIs) have been demonstrated to prevent gastric mucosal injury by mechanisms independent of acid inhibition. Here we demonstrate that both omeprazole and lansoprazole protect human gastric epithelial and endothelial cells against oxidative stress. This effect was abrogated in the presence of the heme oxygenase-1 (HO-1) inhibitor ZnBG. Exposure to either PPI resulted in a strong induction of HO-1 expression on mRNA and protein level, and led to an increased activity of this enzyme. Expression of cyclooxygenase isoforms 1 and 2 remained unaffected, and COX-inhibitors did not antagonize HO-1 induction by PPIs. Our results suggest that the antioxidant defense protein HO-1 is a target of PPIs in both endothelial and gastric epithelial cells. HO-1 induction might account for the gastroprotective effects of PPIs independently of acid inhibition, especially in NSAID gastropathy. Moreover, our findings provide additional perspectives for a possible but yet unexplored use of PPIs in vasoprotection.

  17. Mitochondrial proteomics of the acetic acid - induced programmed cell death response in a highly tolerant Zygosaccharomyces bailii - derived hybrid strain

    PubMed Central

    Guerreiro, Joana F.; Sampaio-Marques, Belém; Soares, Renata; Coelho, Ana V.; Leão, Cecília; Ludovico, Paula; Sá-Correia, Isabel

    2016-01-01

    Very high concentrations of acetic acid at low pH induce programmed cell death (PCD) in both the experimental model Saccharomyces cerevisiae and in Zygosaccharomyces bailii, the latter being considered the most problematic acidic food spoilage yeast due to its remarkable intrinsic resistance to this food preservative. However, while the mechanisms underlying S. cerevisiae PCD induced by acetic acid have been previously examined, the corresponding molecular players remain largely unknown in Z. bailii. Also, the reason why acetic acid concentrations known to be necrotic for S. cerevisiae induce PCD with an apoptotic phenotype in Z. bailii remains to be elucidated. In this study, a 2-DE-based expression mitochondrial proteomic analysis was explored to obtain new insights into the mechanisms involved in PCD in the Z. bailii derived hybrid strain ISA1307. This allowed the quantitative assessment of expression of protein species derived from each of the parental strains, with special emphasis on the processes taking place in the mitochondria known to play a key role in acetic acid - induced PCD. A marked decrease in the content of proteins involved in mitochondrial metabolism, in particular, in respiratory metabolism (Cor1, Rip1, Lpd1, Lat1 and Pdb1), with a concomitant increase in the abundance of proteins involved in fermentation (Pdc1, Ald4, Dld3) was registered. Other differentially expressed identified proteins also suggest the involvement of the oxidative stress response, protein translation, amino acid and nucleotide metabolism, among other processes, in the PCD response. Overall, the results strengthen the emerging concept of the importance of metabolic regulation of yeast PCD. PMID:28357336

  18. MiRNA-194 Regulates Palmitic Acid-Induced Toll-Like Receptor 4 Inflammatory Responses in THP-1 Cells.

    PubMed

    Tian, Huiqun; Liu, Chaoqi; Zou, Xiaohua; Wu, Wei; Zhang, Changcheng; Yuan, Ding

    2015-05-13

    There is strong evidence to suggest that inflammatory responses link obesity and diseases, and the understanding of obesity-induced inflammatory mechanisms is central to the pathogenesis of diseases such asnonalcoholic fatty liver disease(NAFLD) and atherosclerosis that are modified by obesity. Based on this, anti-inflammatory treatments become a potential therapies for obesity-related diseases like NAFLD.A critical role of toll-like receptor (TLR) and its downstream molecules such as tumor necrosis factor receptor-associated factor 6(TRAF6) has been documented in inflammatory response induced by fatty acid. TLR pathway regulation provides a new insight to controlling the inflammatory response induced by fatty acid. Taken together, our study was aimed to understand the mechanism of fatty acid-mediated inflammation and look for an effective target which can prevent the inflammatory response induced by obesity. In this study, we used the saturated fatty acid palmitic acid (PA) to activate TLR4 signal pathway in human monocyte cells THP-1 that established an intracellular inflammatory model. Followed with activated TLR4, downstream molecular TRAF6 was upregulated and ultimately induced proinflammatory cytokine production. Based on this model, we also found that PA downregulated miR-194 expression with TLR4 activation. Moreover, our results showed that key signal molecular TRAF6 is a target of miR-194, overexpression of miR-194 directly decreased TRAF6 expression and attenuated the release of proinflammatory cytokine TNF-α in PA-activated monocyte THP-1. We conclude that miR-194 negatively regulates the TLR4 signal pathway which is activated by PA through directly negative TRAF6 expression.

  19. Docosahexaenoic acid counteracts attenuation of CD95-induced cell death by inorganic mercury

    SciTech Connect

    Gill, Randall; Lanni, Lydia; Jen, K.-L. Catherine; McCabe, Michael J.; Rosenspire, Allen

    2015-01-01

    In the United States the principal environmental exposure to mercury is through dietary consumption of sea food. Although the mechanism by which low levels of mercury affect the nervous system is not well established, epidemiological studies suggest that low level exposure of pregnant women to dietary mercury can adversely impact cognitive development in their children, but that Docosahexaenoic acid (DHA), the most prominent n-polyunsaturated fatty acid (n-PUFA) present in fish may counteract negative effects of mercury on the nervous system. Aside from effects on the nervous system, epidemiological and animal studies have also suggested that low level mercury exposure may be a risk factor for autoimmune disease. However unlike the nervous system where a mechanism linking mercury to impaired cognitive development remains elusive, we have previously suggested a potential mechanism linking low level mercury exposures to immune system dysfunction and autoimmunity. In the immune system it is well established that disruption of CD95 mediated apoptosis leads to autoimmune disease. We have previously shown in vitro as well as in vivo that in lymphocytes burdened with low levels of mercury, CD95 mediated cell death is impaired. In this report we now show that DHA counteracts the negative effect of mercury on CD95 signaling in T lymphocytes. T cells which have been pre-exposed to DHA are able to cleave pro-caspase 3 and efficiently signal programmed cell death through the CD95 signaling pathway, whether or not they are burdened with low levels of mercury. Thus DHA may lower the risk of autoimmune disease after low level mercury exposures. - Highlights: • Inorganic mercury (Hg{sup 2+}) interferes with CD95 mediated cell death in Jurkat T cells • DHA restores the ability of CD95 to signal cell death in Hg{sup 2+} intoxicated T cells • The restoration of CD95 mediated cell death by DHA is correlated with increased activation of Caspase 3.

  20. Eicosapentaenoic acid inhibits TNF-{alpha}-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

    SciTech Connect

    Kim, Hyeon Ho; Lee, Youngae; Eun, Hee Chul Chung, Jin Ho

    2008-04-04

    Eicosapentaenoic acid (EPA) is an omega-3 ({omega}-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-{kappa}B activation induced by tumor necrosis factor (TNF)-{alpha} or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-{alpha}-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-{alpha} induced MMP-9 expression by NF-{kappa}B-dependent pathway. Pretreatment of EPA inhibited TNF-{alpha}-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect I{kappa}B-{alpha} phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-{kappa}B. EPA inhibited TNF-{alpha}-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKK{alpha}-dependent event. Taken together, we demonstrate that EPA inhibits TNF-{alpha}-induced MMP-9 expression through inhibition of p38 and Akt activation.

  1. Retinoic acid induces nuclear FAK translocation and reduces breast cancer cell adhesion through Moesin, FAK, and Paxillin.

    PubMed

    Sanchez, Angel Matías; Shortrede, Jorge Eduardo; Vargas-Roig, Laura María; Flamini, Marina Inés

    2016-07-15

    Breast cancer is the most common malignancy in women, with metastases being the cause of death in 98%. In previous works we have demonstrated that retinoic acid (RA), the main retinoic acid receptor (RAR) ligand, is involved in the metastatic process by inhibiting migration through a reduced expression of the specific migration-related proteins Moesin, c-Src, and FAK. At present, our hypothesis is that RA also acts for short periods in a non-genomic action to cooperate with motility reduction and morphology of breast cancer cells. Here we identify that the administration of 10(-6) M RA (10-20 min) induces the activation of the migration-related proteins Moesin, FAK, and Paxillin in T-47D breast cancer cells. The phosphorylation exerted by the selective agonists for RARα and RARβ, on Moesin, FAK, and Paxillin was comparable to the activation exerted by RA. The RARγ agonist only led to a weak activation, suggesting the involvement of RARα and RARβ in this pathway. We then treated the cells with different inhibitors that are involved in cell signaling to regulate the mechanisms of cell motility. RA failed to activate Moesin, FAK, and Paxillin in cells treated with Src inhibitor (PP2) and PI3K inhibitor (WM), suggesting the participation of Src-PI3K in this pathway. Treatment with 10(-6) M RA for 20 min significantly decreased cell adhesion. However, when cells were treated with 10(-6) M RA and FAK inhibitor, the RA did not significantly inhibit adhesion, suggesting a role of FAK in the adhesion inhibited by RA. By immunofluorescence and immunoblotting analysis we demonstrated that RA induced nuclear FAK translocation leading to a reduced cellular adhesion. These findings provide new information on the actions of RA for short periods. RA participates in cell adhesion and subsequent migration, modulating the relocation and activation of proteins involved in cell migration.

  2. Swelling-activated and arachidonic acid-induced currents are TREK-1 in rat bladder smooth muscle cells

    PubMed Central

    Fukasaku, Mitsuko; Kimura, Junko; Yamaguchi, Osamu

    2016-01-01

    Abstract Using the perforated patch voltage clamp, we investigated swelling-activated ionic channels (SACs) in rat urinary bladder smooth muscle cells. Hypo-osmotic (60%) bath solution increased a membrane current which was inhibited by the SAC inhibitor, gadolinium. The reversal potential of the hypotonicity-induced current shifted in the positive direction by increasing external K+ concentration. The hypotonicity-induced current was inhibited by extracellular acidic pH, phorbol ester and forskolin. These pharmacological properties are identical to those of arachidonic acid-induced current present in these cells, suggesting the presence of TREK-1, a four-transmembrane two pore domain K+ channel. Using RT-PCR we screened rat bladder smooth muscles and cerebellum for expression of TREK-1, TREK-2 and TRAAK mRNAs. Only TREK-1 mRNA was expressed in the bladder, while all three were expressed in the cerebellum. We conclude that a mechanosensitive K+ channel is present in rat bladder myocytes, which is activated by arachidonic acid and most likely is TREK-1. This K+ channel may have an important role in the regulation of bladder smooth muscle tone during urine storage. PMID:26911303

  3. Swelling-activated and arachidonic acid-induced currents are TREK-1 in rat bladder smooth muscle cells.

    PubMed

    Fukasaku, Mitsuko; Kimura, Junko; Yamaguchi, Osamu

    2016-06-08

    Using the perforated patch voltage clamp, we investigated swelling-activated ionic channels (SACs) in rat urinary bladder smooth muscle cells. Hypo-osmotic (60%) bath solution increased a membrane current which was inhibited by the SAC inhibitor, gadolinium. The reversal potential of the hypotonicity-induced current shifted in the positive direction by increasing external K(+) concentration. The hypotonicity-induced current was inhibited by extracellular acidic pH, phorbol ester and forskolin. These pharmacological properties are identical to those of arachidonic acid-induced current present in these cells, suggesting the presence of TREK-1, a four-transmembrane two pore domain K(+) channel. Using RT-PCR we screened rat bladder smooth muscles and cerebellum for expression of TREK-1, TREK-2 and TRAAK mRNAs. Only TREK-1 mRNA was expressed in the bladder, while all three were expressed in the cerebellum. We conclude that a mechanosensitive K(+) channel is present in rat bladder myocytes, which is activated by arachidonic acid and most likely is TREK-1. This K(+) channel may have an important role in the regulation of bladder smooth muscle tone during urine storage.

  4. Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD-1 and suppression by N-acetyl-L-cysteine.

    PubMed

    Aizawa, Shu; Ookawa, Keizou; Kudo, Toshihiro; Asano, Junpei; Hayakari, Makoto; Tsuchida, Shigeki

    2003-10-01

    Since ethacrynic acid (EA), an SH modifier as well as glutathione S-transferase (GST) inhibitor, has been suggested to induce apoptosis in some cell lines, its effects on a human colon cancer cell line DLD-1 were examined. EA enhanced cell proliferation at 20-40 microM, while it caused cell death at 60-100 microM. Caspase inhibitors did not block cell death and DNA ladder formation was not detected. Poly(ADP-ribose) polymerase, however, was cleaved into an 82-kDa fragment, different from an 85-kDa fragment that is specific for apoptosisis. The 82-kDa fragment was not recognized by antibody against PARP fragment cleaved by caspase 3. N-Acetyl-L-cysteine (NAC) completely inhibited EA-induced cell death, but 3(2)-t-butyl-4-hydroxyanisole or pyrrolidinedithiocarbamate ammonium salt did not. Glutathione (GSH) levels were dose-dependently increased in cells treated with EA and this increase was hardly affected by NAC addition. Mitogen-activated protein kinase (MAPK) kinase (MEK) 1, extracellular signal-regulated kinase (ERK) 1 and GST P1-1 were increased in cells treated with 25-75 microM EA, while c-Jun N-terminal kinase (JNK) 1 and p38 MAPK were markedly decreased by 100 microM EA. NAC repressed EA-induced alterations in these MAPKs and GST P1-1. p38 MAPK inhibitors, SB203580 and FR167653, dose-dependently enhanced EA-induced cell death. An MEK inhibitor, U0126, did not affect EA-induced cell death. These studies revealed that EA induced cell death concomitantly with a novel PARP fragmentation, but without DNA fragmentation. p38 MAPK was suggested to play an inhibitory role in EA-induced cell death.

  5. Palmitic Acid-Induced Neuron Cell Cycle G2/M Arrest and Endoplasmic Reticular Stress through Protein Palmitoylation in SH-SY5Y Human Neuroblastoma Cells

    PubMed Central

    Hsiao, Yung-Hsuan; Lin, Ching-I; Liao, Hsiang; Chen, Yue-Hua; Lin, Shyh-Hsiang

    2014-01-01

    Obesity-related neurodegenerative diseases are associated with elevated saturated fatty acids (SFAs) in the brain. An increase in SFAs, especially palmitic acid (PA), triggers neuron cell apoptosis, causing cognitive function to deteriorate. In the present study, we focused on the specific mechanism by which PA triggers SH-SY5Y neuron cell apoptosis. We found that PA induces significant neuron cell cycle arrest in the G2/M phase in SH-SY5Y cells. Our data further showed that G2/M arrest is involved in elevation of endoplasmic reticular (ER) stress according to an increase in p-eukaryotic translation inhibition factor 2α, an ER stress marker. Chronic exposure to PA also accelerates beta-amyloid accumulation, a pathological characteristic of Alzheimer’s disease. Interestingly, SFA-induced ER stress, G2/M arrest and cell apoptosis were reversed by treatment with 2-bromopalmitate, a protein palmitoylation inhibitor. These findings suggest that protein palmitoylation plays a crucial role in SFA-induced neuron cell cycle G2/M arrest, ER stress and apoptosis; this provides a novel strategy for preventing SFA-induced neuron cell dysfunction. PMID:25402647

  6. Repeated citalopram administration counteracts kainic acid-induced spreading of PSA-NCAM-immunoreactive cells and loss of reelin in the adult mouse hippocampus.

    PubMed

    Jaako, Külli; Aonurm-Helm, Anu; Kalda, Anti; Anier, Kaili; Zharkovsky, Tamara; Shastin, Dmitri; Zharkovsky, Alexander

    2011-09-01

    Systemic or intracerebral administration of kainic acid in rodents induces neuronal death followed by a cascade of neuroplastic changes in the hippocampus. Kainic acid-induced neuroplasticity is evidenced by alterations in hippocampal neurogenesis, dispersion of the granule cell layer and re-organisation of mossy fibres. Similar abnormalities are observed in patients with temporal lobe epilepsy and, therefore, kainic acid-induced hippocampal neuroplasticity might mimic pathological mechanisms leading to the formation of 'epileptic brain' in patients with temporal lobe epilepsy. Previous studies have demonstrated that selective serotonin re-uptake inhibitor antidepressants might reduce the severity of seizures in epileptic patients and reduce neuronal death in laboratory animal models of kainic acid-induced neurotoxicity. In the present study, we investigated whether kainic acid-induced neuroplasticity in mice is modulated by the repeated administration of citalopram, a selective serotonin reuptake inhibitor. We found that at the histopathological level, repeated citalopram treatment counteracted the kainic acid-induced neuronal loss and dispersion of young granule neurons expressing the polysialylated neural cell adhesion molecule within the granule cell layer of the hippocampus. Citalopram also counteracted the downregulation of reelin on both mRNA and protein levels induced by kainic acid administration. Our findings indicate that repeated administration of citalopram is able to prevent kainic acid-induced abnormal brain plasticity and thereby prevent the formation of an epileptic phenotype.

  7. PI3K/Akt pathway regulates retinoic acid-induced Hox gene expression in F9 cells.

    PubMed

    Lee, Youra; Lee, Ji-Yeon; Kim, Myoung Hee

    2014-09-01

    Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA-induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA-induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA-induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time-course gene expression profiles for all 39 Hox genes located in four different clusters-Hoxa, Hoxb, Hoxc, and Hoxd-were analyzed. Collinear expression of Hoxa and -b cluster genes was initiated earlier than that of the -c and -d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA-induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.

  8. CD4+ T cell responses in Balb/c mice with food allergy induced by trinitrobenzene sulfonic acid and ovalbumin.

    PubMed

    Sun, Chen-Yi; Bai, Jie; Hu, Tian-Yong; Cheng, Bao-Hui; Ma, Li; Fan, Xiao-Qin; Yang, Ping-Chang; Zheng, Peng-Yuan; Liu, Zhi-Qiang

    2016-06-01

    The rapid increase in atopic diseases is potentially linked to increased hapten exposure, however, the role of haptens in the pathogenesis of food allergy remains unknown. Further studies are required to elucidate the cluster of differentiation 4 positive (CD4+) T cell response to food allergy induced by haptens. Dendritic cells were primed by trinitrobenzene sulfonic acid (TNBS) as a hapten or ovalbumin (OVA) as a model antigen, in a cell culture model. BALB/c mice were sensitized using TNBS and/or OVA. Intestinal Th1/Th2 cell and ovalbumin specific CD4+ T cells proliferation, intestinal cytokines (interleukin‑4 and interferon‑γ) in CD4+ T cells were evaluated. TNBS increased the expression of T cell immunoglobulin and mucin domain‑4 and tumor necrosis factor ligand superfamily member 4 in dendritic cells. Skewed Th2 cell polarization, extensive expression of interleukin‑4, reduced expression of interferon‑γ and forkhead box protein P3 were elicited following concomitant exposure to TNBS and OVA, with reduced regulatory T cells in the mouse intestinal mucosa, whereas a Th1 response was detected when challenged by TNBS or OVA alone. This data suggests that TNBS, as a hapten, combined with food antigens may lead to a Th2 cell response in the intestinal mucosa.

  9. AA-PMe, a novel asiatic acid derivative, induces apoptosis and suppresses proliferation, migration, and invasion of gastric cancer cells.

    PubMed

    Jing, Yue; Wang, Gang; Ge, Ying; Xu, Minjie; Tang, Shuainan; Gong, Zhunan

    2016-01-01

    Asiatic acid (AA; 2α,3β,23-trihydroxyurs-12-ene-28-oic acid) is widely used for medicinal purposes in many Asian countries due to its various bioactivities. A series of AA derivatives has been synthesized in attempts to improve its therapeutic potencies. Herein we investigated the anti-tumor activities of N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe), a novel AA derivative. AA-PMe exhibited a stronger anti-cancer activity than its parent compound AA. AA-PMe inhibited the proliferation of SGC7901 and HGC27 human gastric cancer cells in a dose-dependent manner but had no significant toxicity in human gastric mucosa epithelial cells (GES-1). AA-PMe induced cell cycle arrest in G0/G1 phase and blocked G1-S transition, which correlated well with marked decreases in levels of cyclin D1, cyclin-dependent kinase CKD4, and phosphorylated retinoblastoma protein, and increase in cyclin-dependent kinase inhibitor P15. Further, AA-PMe induced apoptosis of human gastric cancer cells by affecting Bcl-2, Bax, c-Myc, and caspase-3. Moreover, AA-PMe suppressed the migration and invasion of human gastric cancer cells (SGC7901 and HGC27) cells by downregulating the expression of MMP-2 and MMP-9. Overall, this study investigated the potential anti-cancer activities of AA-PMe including inducing apoptosis and suppressing proliferation, migration and invasion of gastric cancer cells, as well as the underlying mechanisms, suggesting that AA-PMe is a promising anti-cancer drug candidate in gastric cancer therapy.

  10. AA-PMe, a novel asiatic acid derivative, induces apoptosis and suppresses proliferation, migration, and invasion of gastric cancer cells

    PubMed Central

    Jing, Yue; Wang, Gang; Ge, Ying; Xu, Minjie; Tang, Shuainan; Gong, Zhunan

    2016-01-01

    Asiatic acid (AA; 2α,3β,23-trihydroxyurs-12-ene-28-oic acid) is widely used for medicinal purposes in many Asian countries due to its various bioactivities. A series of AA derivatives has been synthesized in attempts to improve its therapeutic potencies. Herein we investigated the anti-tumor activities of N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe), a novel AA derivative. AA-PMe exhibited a stronger anti-cancer activity than its parent compound AA. AA-PMe inhibited the proliferation of SGC7901 and HGC27 human gastric cancer cells in a dose-dependent manner but had no significant toxicity in human gastric mucosa epithelial cells (GES-1). AA-PMe induced cell cycle arrest in G0/G1 phase and blocked G1-S transition, which correlated well with marked decreases in levels of cyclin D1, cyclin-dependent kinase CKD4, and phosphorylated retinoblastoma protein, and increase in cyclin-dependent kinase inhibitor P15. Further, AA-PMe induced apoptosis of human gastric cancer cells by affecting Bcl-2, Bax, c-Myc, and caspase-3. Moreover, AA-PMe suppressed the migration and invasion of human gastric cancer cells (SGC7901 and HGC27) cells by downregulating the expression of MMP-2 and MMP-9. Overall, this study investigated the potential anti-cancer activities of AA-PMe including inducing apoptosis and suppressing proliferation, migration and invasion of gastric cancer cells, as well as the underlying mechanisms, suggesting that AA-PMe is a promising anti-cancer drug candidate in gastric cancer therapy. PMID:27073325

  11. Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells.

    PubMed

    Gupta, Sandeep K; Haigh, Brendan J; Seyfert, Hans-Martin; Griffin, Frank J; Wheeler, Thomas T

    2017-03-01

    Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

  12. Neuroprotective effects of docosahexaenoic acid on hippocampal cell death and learning and memory impairments in a valproic acid-induced rat autism model.

    PubMed

    Gao, Jingquan; Wang, Xuelai; Sun, Hongli; Cao, Yonggang; Liang, Shuang; Wang, Han; Wang, Yanming; Yang, Feng; Zhang, Fengyu; Wu, Lijie

    2016-04-01

    Prenatal exposure to valproic acid (VPA) in rat offspring is capable of inducing experimental autism with neurobehavioral aberrations. This study investigated the effect of docosahexaenoic acid (DHA) on hippocampal cell death, learning and memory alteration in an experimental rat autism model. We found that DHA supplementation (75, 150 or 300 mg/kg/day, 21 days) rescued the VPA (600 mg/kg) induced DHA reduction in plasma and hippocampus in a dose-dependent manner, increased the levels of hippocampal p-CaMKII and p-CREB without affecting total protein level, and altered BDNF-AKT-Bcl-2 signaling pathway, as well as inhibited the activity of caspase-3. DHA also influenced the content of malondialdehyde (MDA) and the activities of antioxidant enzymes in the VPA-treated offspring. Consistent with the previous results, we also observed that 300 mg/kg DHA supplementation markedly increased the cell survival, decreased the cell apoptosis, and increased mature neuronal cell in the hippocampus in VPA-treated offspring. Utilizing the Morris water maze test, we found that DHA prevented cognitive impairment in offspring of VPA-treated rats. The data suggested that DHA may play a neuroprotective role in hippocampal neuronal cell and ameliorates dysfunctions in learning and memory in this rat autism model. Thus, DHA could be used as treatment intervention for mitigating behavioral dysfunctions in autism spectrum disorder (ASD).

  13. All-trans-retinoic acid induces integrin-independent B-cell adhesion to ADAM disintegrin domains.

    PubMed

    Bridges, Lance C; Lingo, Joshuah D; Grandon, Rachel A; Kelley, Melissa D

    2008-04-15

    Cell adhesion is an integral aspect of immunity facilitating extravasation of immune cells during homing and activation. All -trans-Retinoic acid ( t-RA) regulates leukocyte differentiation, proliferation, and transmigration. However, the role of t-RA in immune cell adhesion is poorly defined. In this study, we evaluated the impact of t-RA and its metabolism on B and T cell adhesion. Specifically, we address the impact of t-RA on the adhesive properties of the human mature B and T cell lines RPMI 8866, Daudi and Jurkats. The effect of t-RA exposure on cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), a well-established integrin counter receptor involved in immunity, and to nonconventional ADAM integrin ligands was assessed. We show for the first time that t-RA potently induces B cell adhesion in an integrin-independent manner to both VCAM-1 and select ADAM disintegrin domains. Using retinoid extraction and reverse-phase HPLC analysis, we identify the retinoid that is functionally responsible for this augmented adhesion. We also provide evidence that this novel t-RA adhesive response is not prototypical of lymphocytes since both Daudi and Jurkats do not alter their adhesive properties upon t-RA treatment. Further, the t-RA metabolic profiles between these lineages is distinct with 9- cis-retinoic acid being exclusively detected in Jurkat media. This study is the first to demonstrate that t-RA directly induces B cell adhesion in an integrin-independent manner and is not contingent upon t-RA metabolism.

  14. Maslinic Acid Protected PC12 Cells Differentiated by Nerve Growth Factor against β-Amyloid-Induced Apoptosis.

    PubMed

    Yang, Yu-wan; Tsai, Chia-wen; Mong, Mei-chin; Yin, Mei-chin

    2015-12-02

    β-Amyloid peptide (Abeta) was used to induce apoptosis in PC12 cells differentiated by nerve growth factor, and the protective activities of maslinic acid (MA) at 2-16 μM were examined. Abeta treatment lowered Bcl-2 expression, raised Bax expression, and decreased cell viability. MA pretreatments decreased Bax expression, raised the Bcl-2/Bax ratio, and increased cell viability. MA pretreatments retained glutathione content and decreased subsequent Abeta-induced release of reactive oxygen species, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Abeta treatment up-regulated protein expression of p47(phox), gp91(phox), mitogen-activated protein kinase, advanced glycation end product receptor (RAGE), and nuclear factor-κ B (NF-κB). MA pretreatments at 2-16 μM suppressed the expression of proteins including gp91(phox), p47(phox), p-p38, and NF-κB p65, at 4-16 μM down-regulated RAGE and NF-κB p50 expression, and at 8 and 16 μM reduced p-ERK1/2 expression. These novel findings suggest that maslinic acid is a potent compound against Abeta-induced cytotoxicity.

  15. Quinolinic acid induces neuritogenesis in SH-SY5Y neuroblastoma cells independently of NMDA receptor activation.

    PubMed

    Hernandez-Martinez, Juan-Manuel; Forrest, Caroline M; Darlington, L Gail; Smith, Robert A; Stone, Trevor W

    2017-03-01

    Glutamate and nicotinamide adenine dinucleotide (NAD(+) ) have been implicated in neuronal development and several types of cancer. The kynurenine pathway of tryptophan metabolism includes quinolinic acid (QA) which is both a selective agonist at N-methyl-D-aspartate (NMDA) receptors and also a precursor for the formation of NAD(+) . The effect of QA on cell survival and differentiation has therefore been examined on SH-SY5Y human neuroblastoma cells. Retinoic acid (RA, 10 μm) induced differentiation of SH-SY5Y cells into a neuronal phenotype showing neurite growth. QA (50-150 nm) also caused a concentration-dependent increase in the neurite/soma ratio, indicating differentiation. Both RA and QA increased expression of the neuronal marker β3-tubulin in whole-cell homogenates and in the neuritic fraction assessed using a neurite outgrowth assay. Expression of the neuronal proliferation marker doublecortin revealed that, unlike RA, QA did not decrease the number of mitotic cells. QA-induced neuritogenesis coincided with an increase in the generation of reactive oxygen species. Neuritogenesis was prevented by diphenylene-iodonium (an inhibitor of NADPH oxidase) and superoxide dismutase, supporting the involvement of reactive oxygen species. NMDA itself did not promote neuritogenesis and the NMDA antagonist dizocilpine (MK-801) did not prevent quinolinate-induced neuritogenesis, indicating that the effects of QA were independent of NMDA receptors. Nicotinamide caused a significant increase in the neurite/soma ratio and the expression of β3-tubulin in the neuritic fraction. Taken together, these results suggest that QA induces neuritogenesis by promoting oxidizing conditions and affecting the availability of NAD(+) , independently of NMDA receptors.

  16. Knockdown of SALL4 Protein Enhances All-trans Retinoic Acid-induced Cellular Differentiation in Acute Myeloid Leukemia Cells*

    PubMed Central

    Liu, Li; Liu, Liang; Leung, Lai-Han; Cooney, Austin J.; Chen, Changyi; Rosengart, Todd K.; Ma, Yupo; Yang, Jianchang

    2015-01-01

    All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy. PMID:25737450

  17. The bioactive compounds alpha-chaconine and gallic acid in potato extracts decrease survival and induce apoptosis in LNCaP and PC3 prostate cancer cells.

    PubMed

    Reddivari, Lavanya; Vanamala, Jairam; Safe, Stephen H; Miller, J Creighton

    2010-01-01

    We recently reported that colored potato extracts and an anthocyanin rich fraction suppressed lymph-node carcinoma of the prostate (LNCaP) and prostate cancer-3 (PC-3) prostate cancer cell proliferation and induced apoptosis via caspase-dependent and caspase-independent pathways. Chlorogenic acid, caffeic acid, gallic acid, catechin, malvidin, and glycoalkaloids (alpha-chaconine and solanine) have now been identified as the major bioactive components of potato, and their effects on LNCaP and PC-3 cell proliferation and apoptosis have been investigated. alpha-chaconine (5 microg/ml) and gallic acid (15 microg/ml) exhibited potent antiproliferative properties and increased cyclin-dependent kinase inhibitor p27 levels in both cell lines. Both alpha-chaconine and gallic acid induced poly [adenosine diphosphate (ADP)] ribose polymerase cleavage and caspase-dependent apoptosis in LNCaP cells; however, caspase-independent apoptosis through nuclear translocation of endonuclease G was observed in both LNCaP and PC-3 cells. alpha-chaconine and gallic acid activated c-Jun N-terminal protein kinase (JNK), and this response played a major role in induction of caspase-dependent apoptosis in LNCaP cells; whereas modulation of JNK and mitogen-activated protein kinase did not affect alpha-chaconine- and gallic acid-induced caspase-independent apoptosis. These results suggest that apoptosis induced by whole potato extracts in prostate cancer cell lines may be in part due to alpha-chaconine and gallic acid.

  18. Visible light exposure induces VEGF gene expression through activation of retinoic acid receptor-alpha in retinoblastoma Y79 cells.

    PubMed

    Akiyama, Hideo; Tanaka, Toru; Doi, Hiroshi; Kanai, Hiroyoshi; Maeno, Toshitaka; Itakura, Hirotaka; Iida, Tomohiro; Kimura, Yasutaka; Kishi, Shoji; Kurabayashi, Masahiko

    2005-04-01

    Neovascularization of the retina and choroids is the pathological hallmark of many retinopathies, but its molecular mechanisms remain unclear. Vascular endothelial growth factor (VEGF), which is induced by hypoxia or cytokines, plays a critical role in the abnormal growth of blood vessels. In this study, we report that visible light exposure induces VEGF gene expression in retinoblastoma Y79 cells. Fluorescent light exposure (700 lux, wavelength 400 approximately 740 nm) caused a significant increase in VEGF transcripts and protein levels. Such an induction seemed to be specific to certain cells, including photoreceptor cells, because light-induced VEGF expression was not observed in either nontransformed cells, such as retinal pigment epithelium cells, and bovine aortic endothelial cells or transformed cells, such as CV-1 and HepG2 cells. Pertussis toxin and guanosine 5'-[beta-thio]diphosphate, specific inhibitors for rhodopsin-associated G protein, blunted this induction. Progressive deletion and site-specific mutation analyses indicate that light stimulation increases VEGF promoter activity through G+C-rich sequence, which is proven by Sp1 binding sites by supershift assays. Electrophoretic mobility shift assays show that light stimulation increases Sp1 binding. Synthetic retinoic acid receptor-alpha (RARalpha) antagonist completely abrogated light-mediated increase in VEGF expression. Transfection of Y79 cells with dominant negative mutant of RARalpha significantly attenuated the light-mediated induction of VEGF promoter activity. In conclusion, our data indicate that light exposure increases VEGF expression through the mechanisms involving activation of Sp1 and RARalpha signaling in Y79 cells. This study provides new insight into the role of visible light in the transcription and induction of VEGF gene expression.

  19. Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom l-amino acid oxidase.

    PubMed

    Fung, Shin Yee; Lee, Mui Li; Tan, Nget Hong

    2015-03-01

    Snake venom LAAOs have been reported to exhibit a wide range of pharmacological activities, including cytotoxic, edema-inducing, platelet aggregation-inducing/platelet aggregation-inhibiting, bactericidal and antiviral activities. A heat-stable form of l-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom (OH-LAAO) has been shown to exhibit very potent cytotoxicity against human tumorigenic cells but not in their non-tumorigenic counterparts, and the cytotoxicity was due to the apoptosis-inducing effect of the enzyme. In this work, the molecular mechanism of cell death induced by OH-LAAO was investigated. The enzyme exerts its apoptosis-inducing effect presumably via both intrinsic and extrinsic pathways as suggested by the increase in caspase-8 and -9 activities. Oligonucleotide microarray analysis showed that the expression of a total of 178 genes was significantly altered as a result of oxidative stress induced by the hydrogen peroxide generated by the enzyme. Of the 178 genes, at least 27 genes are involved in apoptosis and cell death. These alterations of gene expression was presumably caused by the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidative modifications of signaling molecules that eventually lead to apoptosis and cell death. The very substantial up-regulation of cytochrome P450 genes may also contribute to the potent cytotoxic action of OH-LAAO by producing excessive reactive oxygen species (ROS). In conclusion, the potent apoptosis inducing activity of OH-LAAO was likely due to the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidation of signalling molecules.

  20. RRD-251 enhances all-trans retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells

    PubMed Central

    Bunaciu, Rodica P.; Yen, Andrew

    2016-01-01

    All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest of HL-60 cells. Responding to an RA-induced cytosolic signaling machine, c-Raf translocates to the nucleus, providing propulsion for RA-induced differentiation. This novel mechanism is not understood, but presumably reflects c-Raf binding with nuclear gene regulatory proteins. RRD-251 is a small molecule that prevents the interaction of c-Raf and RB, the retinoblastoma tumor suppressor protein. The involvement of c-Raf and RB in RA-induced differentiation motivates interest in the effects of combined RA and RRD-251 treatment on leukemic cell differentiation. We demonstrate that RRD-251 enhances RA-induced differentiation. Mechanistically, we find that nuclear translocated c-Raf associates with pS608 RB. RA causes loss of pS608 RB, where cells with hypophosphorylated S608 RB are G0/G1 restricted. Corroborating the pS608 RB hypophosphorylation, RB sequestration of E2F increased with concomitant loss of cdc6 expression, which is known to be driven by E2F. Hypophosphorylation of S608 RB releases c-Raf from RB sequestration to bind other nuclear targets. Release of c-Raf from RB sequestration results in enhanced association with GSK-3 which is phosphorylated at its S21/9 inhibitory sites. c-Raf binding to GSK-3 is associated with dissociation of GSK-3 and RARα, thereby relieving RARα of GSK-3 inhibition. RRD-251 amplifies each of these RA-induced events. Consistent with the posited enhancement of RARα transcriptional activity by RRD-251, RRD-251 increases the RARE-driven CD38 expression per cell. The RA/c-Raf/GSK-3/RARα axis emerges as a novel differentiation regulatory mechanism susceptible to RRD-251, suggesting enhancing RA-effects with RRD-251 in therapy. PMID:27331409

  1. Palmitic acid-induced apoptosis in pancreatic β-cells is increased by liver X receptor agonist and attenuated by eicosapentaenoate.

    PubMed

    Liang, Huasheng; Zhong, Yuhua; Zhou, Shaobi; Li, Qingdi Quentin

    2011-01-01

    Saturated fatty acids are implicated in the development of diabetes via the impairment of pancreatic islet β-cell viability and function. Liver X receptors (LXRs) and eicosapentaenoate (EPA) are known regulators of fatty acid metabolism. However, their roles in the pathogenesis of diabetes remain incompletely understood. The aim of this study was to determine the effects of EPA and the LXR agonist T0901317 on saturated fatty acid (palmitic acid)-induced apoptosis in the insulinoma β-cell line INS-1, a model for insulin-secreting β-cells. T0901317 significantly promoted palmitic acid-induced apoptotic cell death in the INS-1 cells. Consistent with these results, caspase-3 activity and BAX and sterol regulatory element binding protein-1c (SREBP-1c) mRNA levels were markedly increased in INS-1 cells co-administered palmitic acid and T0901317. The production of reactive oxygen species was considerably higher in the cells cultured concurrently with T0901317 and palmitic acid than in the cells incubated with either agent alone. EPA treatment attenuated the cellular death promoted by palmitic acid and T0901317 in the INS-1 cells, disclosing a possible mediating mechanism involving the inhibition of SREBP-1c. Finally, T0901317 up-regulated the palmitic acid-induced expression of p27(KIP1), transforming growth factor beta 1, and SMAD3 proteins in INS-1 cells. These results demonstrate that palmitic acid-induced apoptosis in β-cells is enhanced by T0901317 via the activation of LXRs and is blocked by EPA via the inhibition of SREBP-1c, suggesting that the regulation of lipogenesis and lipotoxicity affecting pancreatic β-cell viability and insulin production may be a unique strategy for diabetes therapy.

  2. Selenite promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells

    PubMed Central

    Misra, Sougat; Selvam, Arun Kumar; Wallenberg, Marita; Ambati, Aditya; Matolcsy, András; Magalhaes, Isabelle; Lauter, Gilbert; Björnstedt, Mikael

    2016-01-01

    Selective targeting of the PML/RARα oncoprotein demonstrates a successful molecular targeted therapy in acute promyelocytic leukemia (APL) with a typical t(15:17) chromosomal translocation. The zinc-thiolate coordination is critical for structural stability of zinc finger proteins, including the PML moiety of PML/RARα. Based on the known interaction of redox-active selenium compounds with thiolate ligands of zinc, we herein have investigated the abrogatory effects of selenite alone or in combination with all-trans retinoic acid on PML/RARα and the possible effects on differentiation in these cells. At pharmacological concentrations, selenite inhibited the proliferation and survival of APL originated NB4 cells. In combination with ATRA, it potentiated the differentiation of NB4 cells without any differentiating effects of its own as a single agent. Concordant with our hypothesis, PML/RARα oncoprotein expression was completely abrogated by selenite. Increased expression of RAR, PU.1 and FOXO3A transcription factors in the combined treatment suggested the plausible basis for increased differentiation in these cells. We show that selenite at clinically achievable dose targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemic cells in combination with ATRA. The present investigation reveals the hitherto unknown potential of selenite in targeted abrogation of PML/RARα in APL cells with prospective therapeutic value. PMID:27732960

  3. Oxidative stress-driven mechanisms of nordihydroguaiaretic acid-induced apoptosis in FL5.12 cells

    SciTech Connect

    Deshpande, Vaidehee S. . E-mail: vaidehee@hotmail.com; Kehrer, James P.

    2006-08-01

    Nordihydroguaiaretic acid (NDGA), a general lipoxygenase (LOX) enzyme inhibitor, induces apoptosis independently of its activity as a LOX inhibitor in murine pro-B lymphocytes (FL.12 cells) by a mechanism that is still not fully understood. Glutathione depletion, oxidative processes and mitochondrial depolarization appear to contribute to the apoptosis induced by NDGA. The current data demonstrate that NDGA (20 {mu}M)-induced apoptosis in FL5.12 cells is partially protected by N-acetylcysteine (NAC) (10 mM) and dithiothreitol (DTT) (500 {mu}M) pretreatment, confirming a role for oxidative processes. In addition, the treatment of FL5.12 cells with NDGA led to an increase in phosphorylation and activation of the MAP kinases ERK, JNK and p38. Although pretreatment with ERK inhibitors (PD98059 or U0126) abolished ERK phosphorylation in response to NDGA, neither inhibitor had any effect on NDGA-induced apoptosis. SP600125, a JNK inhibitor, did not have any effect on NDGA-induced phosphorylation of JNK nor apoptosis. Pretreatment with the p38 inhibitor SB202190 attenuated NDGA-induced apoptosis by 30% and also abolished p38 phosphorylation, compared to NDGA treatment alone. NAC, but not DTT, also decreased the phosphorylation of p38 and JNK supporting a role for oxidative processes in activating these kinases. Neither NAC nor DTT blocked the phosphorylation of ERK suggesting that this activation is not related to oxidative stress. The release of cytochrome c and activation of caspase-3 induced by NDGA were inhibited by NAC. SB202190 slightly attenuated caspase-3 activation and had no effect on the release of cytochrome c. These data suggest that several independent mechanisms, including oxidative reactions, activation of p38 kinase and cytochrome c release contribute to NDGA-induced apoptosis.

  4. Ursolic acid from Trailliaedoxa gracilis induces apoptosis in medullary thyroid carcinoma cells

    PubMed Central

    AGUIRIANO-MOSER, VICTOR; SVEJDA, BERNHARD; LI, ZENG-XIA; STURM, SONJA; STUPPNER, HERMANN; INGOLIC, ELISABETH; HÖGER, HARALD; SIEGL, VERONIKA; MEIER-ALLARD, NATHALIE; SADJAK, ANTON; PFRAGNER, ROSWITHA

    2015-01-01

    Medullary thyroid carcinoma (MTC) originates from the C-cells of the thyroid and is not sensitive to radiation or chemotherapy. Therefore, surgical removal of the tumor tissue in its entirety is the only curative treatment for MTC. The present study aimed to examine the potential mechanisms of action of extracts of Trailliaedoxa gracilis (TG; WW Smith & Forrest), a plant from the province of Sichuan, China, and of ursolic acid (UA), a pentacyclic triterpen present in TG, on the MTC-SK MTC cell line. A total of 13 TG fractions and UA were examined in vitro for their effects on cell morphology, cell number, proliferation and rates of apoptosis. Reverse transcription-quantitative polymerase chain reaction of nuclear factor-κB essential modifier (NEMO) was performed to delineate the role of the apoptotic pathway following treatment with UA. TG and UA were examined in vivo in xenotransplanted MTC-bearing severe combined immunodeficient mice. The TG fractions exhibited antiproliferative effects, with inhibition of mitochondrial activity in the tumor cells at concentrations, which caused no impairment of the normal control cells. The apoptotic rates of the MTC-SK cells treated with the TG fractions and UA were determined, in which no marked tumor inhibition was observed in the treated MTC-mice, and no change in the expression of NEMO was detected in the treated MTC-SK cells. The observation of early-onset activation of caspase 8 suggested that the responsible factor was linked to NEMO, an anti-apoptotic protein. However, no differences in the mRNA transcription levels of NEMO were detected in MTC-SK cells treated with UA, suggesting that this protein was not associated with the signal transducer and activator of transcription 3 pathway. PMID:26151624

  5. Formation and accumulation of protoporphyrin IX in tumor and nontumor cell lines induced by 5-aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Fernandez, Sandra R.; Milanetto, Marilia; Bagnato, Vanderlei S.; Imasato, Hidetake; Perussi, Janice R.

    2005-04-01

    The endogenous photosensitizer 5-aminolevulinic acid (ALA) is a haem precursor and induces the synthesis of protoporphyrin IX (PpIX) in mitochondria-containing cells. Due to the slow conversion of porphyrins to haem, high levels of PPIX are found in the tissues, sufficient to produce a photodynamic effect following exposure to light. Since PpIX accumulates effectively in tumor cells, the use of ALA leads to a better photoselectivity than Photofrin. However, this selectivity has not been sufficiently studied. As far as we know there is just one study comparing the amount of accumulated PpIX in non-tumor and tumor cell lines. In this work we attempt to compare not just the production but also the accumulation and cytotoxicity of PpIX in non-tumor (VERO) versus tumor (Hep-2) cells induced by the use of ALA. The results have shown that both non-tumor and tumor cell lines produce the same amount of PpIX but just the tumor cells can accumulate PpIX. So, under illumination, only the tumor cells will be killed.

  6. Caveolin-1 plays a key role in the oleanolic acid-induced apoptosis of HL-60 cells.

    PubMed

    Ma, Wei; Wang, Di-Di; Li, Li; Feng, Yu-Kuan; Gu, Hong-Mei; Zhu, Gui-Ming; Piao, Jin-Hua; Yang, Yu; Gao, Xu; Zhang, Peng-Xia

    2014-07-01

    Our previous study found that caveolin-1 (CAV-1) protein expression is upregulated during oleanolic acid (OA)-induced inhibition of proliferation and promotion of apoptosis in HL-60 cells. CAV-1 is the main structural protein component of caveolae, playing important roles in tumorigenesis and tumor development. It has been shown that cav-1 expression is lower in leukemia cancer cell lines SUP-B15, HL-60, THP-1 and K562 and in chronic lymphocytic leukemia primary (CLP) cells when compared with normal white blood cells, with the lowest cav-1 expression level found in HL-60 cells. To study the effects of cav-1 in HL-60 cells and the effects of cav-1 overexpression on OA drug efficacy, cav-1 was overexpressed in HL-60 cells using lentiviral-mediated transfection combined with OA treatment. The results showed that cav-1 overexpression inhibited HL-60 cell proliferation, promoted apoptosis, arrested the cell cycle in the G1 phase and inhibited activation of the PI3K/AKT/mTOR signaling pathway. Overexpression of CAV-1 also increased HL-60 cell sensitivity to OA. To further verify whether OA affects HL-60 cells via the activation of downstream signaling pathways by CAV-1, cav-1 gene expression was silenced using RNAi, and the cells were treated with OA to examine its efficacy. The results showed that after cav-1 silencing, OA had little effect on cell activity, apoptosis, the cell cycle and phosphorylation of HL-60 cells. This study is the first to show that CAV-1 plays a crucial role in the effects of OA on HL-60 cells.

  7. PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells.

    PubMed

    Barque, J P; Lagaye, S; Ladoux, A; Della Valle, V; Abita, J P; Larsen, C J

    1987-09-30

    PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.

  8. Suberoylanilide hydroxamic acid (SAHA) inhibits EGF-induced cell transformation via reduction of cyclin D1 mRNA stability

    SciTech Connect

    Zhang, Jingjie; Ouyang, Weiming; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wang, York; Li, Xuejun; Huang, Chuanshu

    2012-09-01

    Suberoylanilide hydroxamic acid (SAHA) inhibiting cancer cell growth has been associated with its downregulation of cyclin D1 protein expression at transcription level or translation level. Here, we have demonstrated that SAHA inhibited EGF-induced Cl41 cell transformation via the decrease of cyclin D1 mRNA stability and induction of G0/G1 growth arrest. We found that SAHA treatment resulted in the dramatic inhibition of EGF-induced cell transformation, cyclin D1 protein expression and induction of G0/G1 growth arrest. Further studies showed that SAHA downregulation of cyclin D1 was only observed with endogenous cyclin D1, but not with reconstitutionally expressed cyclin D1 in the same cells, excluding the possibility of SAHA regulating cyclin D1 at level of protein degradation. Moreover, SAHA inhibited EGF-induced cyclin d1 mRNA level, whereas it did not show any inhibitory effect on cyclin D1 promoter-driven luciferase reporter activity under the same experimental conditions, suggesting that SAHA may decrease cyclin D1 mRNA stability. This notion was supported by the results that treatment of cells with SAHA decreased the half-life of cyclin D1 mRNA from 6.95 h to 2.57 h. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR expression, which has been well-characterized as a positive regulator of cyclin D1 mRNA stability. Thus, our study identifies a novel mechanism responsible for SAHA inhibiting cell transformation via decreasing cyclin D1 mRNA stability and induction of G0/G1 growth arrest in Cl41 cells. -- Highlights: ► SAHA inhibits cell transformation in Cl41 cells. ► SAHA suppresses Cyclin D1 protein expression. ► SAHA decreases cyclin D1 mRNA stability.

  9. Effects of cyclopiazonic acid and dexamethasone on serotonin-induced calcium responses in vascular smooth muscle cells.

    PubMed

    Selli, Cigdem; Tosun, Metiner

    2016-06-01

    We previously observed that sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) blockade by cyclopiazonic acid (CPA) significantly potentiates serotonin (5-hydroxytryptamine (5-HT))-induced vascular contractions. Furthermore, 5-HT receptor antagonist methysergide partially inhibited CPA-potentiated 5-HT contractions. In the present study, we further investigated whether SERCA inhibition potentiates 5-HT-induced Ca(2+) responses along with attenuating the receptor antagonism by store-operated Ca(2+) (SOC) entry and protein kinase C (PKC)-mediated mechanisms. The effects of dexamethasone that was previously shown to induce SOC entry and enhance 5-HT responses were also tested. For this purpose, intracellular Ca(2+) levels were monitored in A7r5 embryonic rat vascular smooth muscle cells by spectrofluorometry using the fluorescent indicator fura-2. The results showed that CPA, although not dexamethasone, significantly potentiated 5-HT-induced Ca(2+) elevations. Ketanserin partially decreased 5-HT-induced and CPA-potentiated Ca(2+) elevations whereas both PKC inhibitor D-sphingosine and SOC entry blocker 2-aminoethoxydiphenyl borate (2-APB) abolished the remaining responses. The data suggests that diminished antagonistic effect on 5-HT-induced Ca(2+) elevations in the presence of SERCA inhibition is induced by SOC entry and PKC activation.

  10. Lysophosphatidic acid-induced ADAM12 expression mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth.

    PubMed

    Do, Eun Kyoung; Kim, Young Mi; Heo, Soon Chul; Kwon, Yang Woo; Shin, Sang Hun; Suh, Dong-Soo; Kim, Ki-Hyung; Yoon, Man-Soo; Kim, Jae Ho

    2012-11-01

    Lysophosphatidic acid (LPA) is involved in mesenchymal stem cell-stimulated tumor growth in vivo. However, the molecular mechanism by which mesenchymal stem cells promote tumorigenesis remains elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induced the expression of ADAM12, a disintegrin and metalloproteases family member, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated ADAM12 expression was abrogated by pretreatment of hASCs with the LPA receptor 1 inhibitor Ki16425 or by small interfering RNA-mediated silencing of LPA receptor 1, suggesting a key role for the LPA-LPA receptor 1 signaling axis in A549 CM-stimulated ADAM12 expression. Silencing of ADAM12 expression using small interfering RNA or short hairpin RNA abrogated LPA-induced expression of both α-smooth muscle actin, a marker of carcinoma-associated fibroblasts, and ADAM12 in hASCs. Using a xenograft transplantation model of A549 cells, we demonstrated that silencing of ADAM12 inhibited the hASC-stimulated in vivo growth of A549 xenograft tumors and the differentiation of transplanted hASCs to α-smooth muscle actin-positive carcinoma-associated fibroblasts. LPA-conditioned medium from hASCs induced the adhesion of A549 cells and silencing of ADAM12 inhibited LPA-induced expression of extracellular matrix proteins, periostin and βig-h3, in hASCs and LPA-conditioned medium-stimulated adhesion of A549 cells. These results suggest a pivotal role for LPA-stimulated ADAM12 expression in tumor growth and the differentiation of hASCs to carcinoma-associated fibroblasts expressing α-smooth muscle actin, periostin, and βig-h3.

  11. Influence of carboxylic acid functionalization on the cytotoxic effects induced by single wall carbon nanotubes on human endothelial cells (HUVEC).

    PubMed

    Gutiérrez-Praena, Daniel; Pichardo, Silvia; Sánchez, Elena; Grilo, Antonio; Cameán, Ana Maria; Jos, Angeles

    2011-12-01

    A vast variety of nanomaterials have been developed in the recent years, being carbon nanotubes (CNTs) the ones that have attracted more attention, due to its unique properties which make them suitable for numerous applications. Consequently, it is predicted that tons of CNTs will be produced worldwide every year, being its exposure of toxicological concern. Nanomaterials, once into the body, can translocate from the uptake sites to the blood circulation or the lymphatic system, resulting in distribution throughout the body. Thus, the vascular endothelium can be in contact with them and can suffer from their toxic effects. In this regard, the aim of this work was to investigate the cytotoxicity of single-walled carbon nanotubes (SWCNTs) on human endothelial cells evaluating the influence of acid carboxylic functionalization and also the exposure time (24 and 48 h). Biomarkers assessed were neutral red uptake, protein content, a tetrazolium salt metabolization and cell viability by means of the Trypan blue exclusion test. Cells were exposed to concentrations between 0 and 800 μg/mL SWCNTs for 24 and 48 h. Results have shown that both SWCNTs and carboxylic acid functionalized single-walled carbon nanotubes (COOH-SWCNTs) induce toxic effects in HUVEC cells in a concentration- and time-dependent way. Moreover, the carboxylic acid functionalization results in a higher toxicity compared to the SWCNTs.

  12. Leucine-Rich Repeat Kinase 2 Modulates Retinoic Acid-Induced Neuronal Differentiation of Murine Embryonic Stem Cells

    PubMed Central

    Schulz, Cathrin; Paus, Marie; Frey, Katharina; Schmid, Ramona; Kohl, Zacharias; Mennerich, Detlev; Winkler, Jürgen; Gillardon, Frank

    2011-01-01

    Background Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. Methodology/Principal Findings In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/− cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. Conclusion/Significance Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases. PMID:21695257

  13. Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells.

    PubMed

    Rzeski, Wojciech; Stepulak, Andrzej; Szymański, Marek; Sifringer, Marco; Kaczor, Józef; Wejksza, Katarzyna; Zdzisińska, Barbara; Kandefer-Szerszeń, Martyna

    2006-10-01

    Betulinic acid (BA) is a pentacyclic triterpene found in many plant species, among others in the bark of white birch Betula alba. BA was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial and anti-inflammatory activities, and in particular to inhibit growth of cancer cells. The aim of the study was further in vitro characterization of BA anticancer activity. In this study, we demonstrated a remarkable antiproliferative effect of BA in all tested tumor cell cultures including neuroblastoma, rabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukemia and multiple myeloma, as well as in primary cultures isolated from ovarian carcinoma, cervical carcinoma and glioblastoma multiforme. Furthermore, we have shown that BA decreased cancer cell motility and induced apoptotic cell death. We also observed decrease of bcl2 and cyclin D1 genes expression, and increase of bax gene expression after betulinic acid treatment. These findings demonstrate the anticancer potential of betulinic acid and suggest that it may be taken into account as a supportive agent in the treatment of cancers with different tissue origin.

  14. Palmitic acid induces interleukin-1β secretion via NLRP3 inflammasomes and inflammatory responses through ROS production in human placental cells.

    PubMed

    Shirasuna, Koumei; Takano, Hiroki; Seno, Kotomi; Ohtsu, Ayaka; Karasawa, Tadayoshi; Takahashi, Masafumi; Ohkuchi, Akihide; Suzuki, Hirotada; Matsubara, Shigeki; Iwata, Hisataka; Kuwayama, Takehito

    2016-08-01

    Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1β secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1β release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1β secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1β, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1β, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications.

  15. Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures

    PubMed Central

    Rodas-Junco, Beatriz A; Cab-Guillen, Yahaira; Muñoz-Sanchez, J Armando; Vázquez-Flota, Felipe; Monforte-Gonzalez, Miriam; Hérnandez-Sotomayor, S M Teresa

    2013-01-01

    Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

  16. Identification of Immunopotentiating Lactic Acid Bacteria that Induce Antibody Production by in vitro Stimulated Human Peripheral Blood Mononuclear Cells.

    PubMed

    Yamashita, Makiko; Hitaka, Akira; Fujino, Himiko; Matsumoto, Takashi; Hasegawa, Takanori; Morimatsu, Fumiki; Fujiki, Tsukasa; Katakura, Yoshinori

    2012-01-01

    L-leucyl-L-leucine methyl ester (LLME) is known to remove lysosome-rich cells from human peripheral blood mononuclear cells (PBMCs). To evaluate the immunopotentiating ability of lactic acid bacteria (LAB), we adopted the in vitro stimulation protocol of LLME-treated PBMCs as a model assay system and monitored the level of antibody produced by stimulated PBMCs. The results indicated that several LAB strains have immunopotentiating ability against PBMCs, as evidenced by the enhanced antibody production and increased number of antigen-specific B cells. Next, we identified T cells as the direct target cells of the immunopotentiating LAB strain L32, suggesting that L32 induced antibody production by PBMCs through T-cell activation. Finally, we tested the immunopotentiating ability of ligands for Toll-like receptor 2 (TLR2), which is known to mediate the LAB signal, and observed that both L32 and one of the TLR2 ligands, LTA-BS, induced antigen-specific antibody production by in vitro stimulated PBMC. This suggests that L32 and LTA-BS can be used as an adjuvant for stimulating immune reaction in PBMCs.

  17. Methylseleninic acid (MSA) inhibits 17β-estradiol-induced cell growth in breast cancer T47D cells via enhancement of the antioxidative thioredoxin/ thioredoxin reductase system.

    PubMed

    Okuno, Tomofumi; Miura, Kiyoshi; Sakazaki, Fumitoshi; Nakamuro, Katsuhiko; Ueno, Hitoshi

    2012-01-01

    The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17β-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.

  18. Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice.

    PubMed

    Sakamoto, Kenji; Murakami, Yuta; Sawada, Shohei; Ushikubo, Hiroko; Mori, Asami; Nakahara, Tsutomu; Ishii, Kunio

    2016-11-15

    Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca(2+) influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo. The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp(1)]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp(1)]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4H-pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo.

  19. Cell signaling in NMDA preconditioning and neuroprotection in convulsions induced by quinolinic acid.

    PubMed

    Severino, Patricia Cardoso; Muller, Gabriele do Amaral Silva; Vandresen-Filho, Samuel; Tasca, Carla Inês

    2011-10-10

    The search for novel, less invasive therapeutic strategies to treat neurodegenerative diseases has stimulated scientists to investigate the mechanisms involved in preconditioning. Preconditioning has been report to occur in many organs and tissues. In the brain, the modulation of glutamatergic transmission is an important and promising target to the use of effective neuroprotective agents. The glutamatergic excitotoxicity is a factor common to neurodegenerative diseases and acute events such as cerebral ischemia, traumatic brain injury and epilepsy. In this review we focus on the neuroprotection and preconditioning by chemical agents. Specially, chemical preconditioning models using N-methyl-d-aspartate (NMDA) pre-treatment, which has demonstrated to lead to neuroprotection against seizures and damage to neuronal tissue induced by quinolinic acid (QA). Here we attempted to gather important results obtained in the study of cellular and molecular mechanisms involved in NMDA preconditioning and neuroprotection.

  20. Pyruvate remediation of cell stress and genotoxicity induced by haloacetic acid drinking water disinfection by-products.

    PubMed

    Dad, Azra; Jeong, Clara H; Pals, Justin A; Wagner, Elizabeth D; Plewa, Michael J

    2013-10-01

    Monohaloacetic acids (monoHAAs) are a major class of drinking water disinfection by-products (DBPs) and are cytotoxic, genotoxic, mutagenic, and teratogenic. We propose a model of toxic action based on monoHAA-mediated inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a target cytosolic enzyme. This model predicts that GAPDH inhibition by the monoHAAs will lead to a severe reduction of cellular ATP levels and repress the generation of pyruvate. A loss of pyruvate will lead to mitochondrial stress and genomic DNA damage. We found a concentration-dependent reduction of ATP in Chinese hamster ovary cells after monoHAA treatment. ATP reduction per pmol monoHAA followed the pattern of iodoacetic acid (IAA) > bromoacetic acid (BAA) > chloroacetic acid (CAA), which is the pattern of potency observed with many toxicological endpoints. Exogenous supplementation with pyruvate enhanced ATP levels and attenuated monoHAA-induced genomic DNA damage as measured with single cell gel electrophoresis. These data were highly correlated with the SN 2 alkylating potentials of the monoHAAs and with the induction of toxicity. The results from this study strongly support the hypothesis that GAPDH inhibition and the possible subsequent generation of reactive oxygen species is linked with the cytotoxicity, genotoxicity, teratogenicity, and neurotoxicity of these DBPs.

  1. Valproic acid inhibits irradiation-induced epithelial-mesenchymal transition and stem cell-like characteristics in esophageal squamous cell carcinoma

    PubMed Central

    Kanamoto, Ayako; Ninomiya, Itasu; Harada, Shinichi; Tsukada, Tomoya; Okamoto, Koichi; Nakanuma, Shinichi; Sakai, Seisho; Makino, Isamu; Kinoshita, Jun; Hayashi, Hironori; Oyama, Katsunobu; Miyashita, Tomoharu; Tajima, Hidehiro; Takamura, Hiroyuki; Fushida, Sachio; Ohta, Tetsuo

    2016-01-01

    Esophageal carcinoma is one of the most aggressive malignancies, and is characterized by poor response to current therapy and a dismal survival rate. In this study we investigated whether irradiation induces epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC) TE9 cells and whether the classic histone deacetylase (HDAC) inhibitor valproic acid (VPA) suppresses these changes. First, we showed that 2 Gy irradiation induced spindle cell-like morphologic changes, decreased expression of membranous E-cadherin, upregulated vimentin expression, and altered the localization of β-catenin from its usual membrane-bound location to cytoplasm in TE9 cells. Irradiation induced upregulation of transcription factors including Slug, Snail, and Twist, which regulate EMT. Stimulation by irradiation resulted in increased TGF-β1 and HIF-1α expression and induced Smad2 and Smad3 phosphorylation. Furthermore, irradiation enhanced CD44 expression, indicating acquisition of cancer stem-like cell properties. In addition, irradiation enhanced invasion and migration ability with upregulation of matrix metalloproteinases. These findings indicate that single-dose irradiation can induce EMT in ESCC cells. Second, we found that treatment with 1 mM VPA induced reversal of EMT caused by irradiation in TE9 cells, resulting in attenuated cell invasion and migration abilities. These results suggest that VPA might have clinical value to suppress irradiation-induced EMT. The reversal of EMT by HDAC inhibitors may be a new therapeutic strategy to improve the effectiveness of radiotherapy in ESCC by inhibiting the enhancement of invasion and metastasis. PMID:27826618

  2. Docosahexaenoic acid counteracts attenuation of CD95-induced cell death by inorganic mercury.

    PubMed

    Gill, Randall; Lanni, Lydia; Jen, K-L Catherine; McCabe, Michael J; Rosenspire, Allen

    2015-01-01

    In the United States the principal environmental exposure to mercury is through dietary consumption of sea food. Although the mechanism by which low levels of mercury affect the nervous system is not well established, epidemiological studies suggest that low level exposure of pregnant women to dietary mercury can adversely impact cognitive development in their children, but that Docosahexaenoic acid (DHA), the most prominent n-polyunsaturated fatty acid (n-PUFA) present in fish may counteract negative effects of mercury on the nervous system. Aside from effects on the nervous system, epidemiological and animal studies have also suggested that low level mercury exposure may be a risk factor for autoimmune disease. However unlike the nervous system where a mechanism linking mercury to impaired cognitive development remains elusive, we have previously suggested a potential mechanism linking low level mercury exposures to immune system dysfunction and autoimmunity. In the immune system it is well established that disruption of CD95 mediated apoptosis leads to autoimmune disease. We have previously shown in vitro as well as in vivo that in lymphocytes burdened with low levels of mercury, CD95 mediated cell death is impaired. In this report we now show that DHA counteracts the negative effect of mercury on CD95 signaling in T lymphocytes. T cells which have been pre-exposed to DHA are able to cleave pro-caspase 3 and efficiently signal programmed cell death through the CD95 signaling pathway, whether or not they are burdened with low levels of mercury. Thus DHA may lower the risk of autoimmune disease after low level mercury exposures.

  3. Gallic Acid Inhibits Proliferation and Induces Apoptosis in Lymphoblastic Leukemia Cell Line (C121)

    PubMed Central

    Sourani, Zahra; Pourgheysari, Batoul; Beshkar, Pezhman; Shirzad, Hedayatollah; Shirzad, Moein

    2016-01-01

    Leukemia is known as the world’s fifth most prevalent cancer. New cytotoxic drugs have created considerable progress in the treatment, but side effects are still the important cause of mortality. Plant derivatives have been recently considered as important sources for the treatment of various diseases, including cancer. Gallic acid (GA) is a polyhydroxyphenolic compound with a wide range of biological functions. The aim of the present study was to evaluate the effect of GA on proliferation inhibition and apoptosis induction of a lymphoblastic leukemia cell line. Jurkat cell (C121) line was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) with different concentrations of GA (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM) for 24, 48 and 72 hours. The effect of GA on cell viability was measured using MTS assay. Induction of apoptosis was evaluated with Annexin V-FITC/PI kit and flow cytometry. Data were analyzed by SPSS version 20 using Kruskal-Wallis and Dunn’s multiple comparison tests. Decline of cell viability to less than 50% was observed at 60.3±1.6, 50.9±1.5, and 30.9±2.8 μM concentration after 24, 48, and 72 hours incubation, respectively. All concentrations of GA (10, 30, 50 and 80 μM) enhanced apoptosis compared to the control (P<0.05). The results demonstrate that the polyphenolic compound, GA, is effective in inhibition of proliferation and induction of apoptosis in Jurkat cell line. It is recommended to study the mechanism of apoptosis induction in future investigations. PMID:27853333

  4. Salvianolic Acid B Inhibits Hydrogen Peroxide-Induced Endothelial Cell Apoptosis through Regulating PI3K/Akt Signaling

    PubMed Central

    Liu, Chen-Li; Xie, Li-Xia; Li, Min; Durairajan, Siva Sundara Kumar; Goto, Shinya; Huang, Jian-Dong

    2007-01-01

    Background Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H2O2) is implicated in the pathogenesis of cerebrovascular disorders. Methodology and Principal Findings By examining the effect of Sal B on H2O2-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H2O2-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H2O2 induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H2O2 and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H2O2-induced apoptosis, suggesting that Sal B prevents H2O2-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway. Significance Our findings provide the first evidence that H2O2 induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H2O2-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway. PMID:18091994

  5. Agmatine protects Müller cells from high-concentration glucose-induced cell damage via N-methyl-D-aspartic acid receptor inhibition.

    PubMed

    Han, Ning; Yu, Li; Song, Zhidu; Luo, Lifu; Wu, Yazhen

    2015-07-01

    Neural injury is associated with the development of diabetic retinopathy. Müller cells provide structural and metabolic support for retinal neurons. High glucose concentrations are known to induce Müller cell activity. Agmatine is an endogenous polyamine, which is enzymatically formed in the mammalian brain and has exhibited neuroprotective effects in a number of experimental models. The aims of the present study were to investigate whether agmatine protects Müller cells from glucose-induced damage and to explore the mechanisms underlying this process. Lactate dehydrogenase activity and tumor necrosis factor-α mRNA expression were significantly reduced in Müller cells exposed to a high glucose concentration, following agmatine treatment, compared with cells not treated with agmatine. In addition, agmatine treatment inhibited glucose-induced Müller cell apoptosis, which was associated with the regulation of Bax and Bcl-2 expression. Agmatine treatment suppressed glucose-induced phosphorylation of mitogen-activated protein kinase (MAPK) protein in Müller cells. The present study demonstrated that the protective effects of agmatine on Müller cells were inhibited by N-methyl-D-aspartic acid (NMDA). The results of the present study suggested that agmatine treatment protects Müller cells from high-concentration glucose-induced cell damage. The underlying mechanisms may relate to the anti-inflammatory and antiapoptotic effects of agmatine, as well as to the inhibition of the MAPK pathway, via NMDA receptor suppression. Agmatine may be of use in the development of novel therapeutic approaches for patients with diabetic retinopathy.

  6. Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells.

    PubMed

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Acierno, Giovanni; Andolfo, Immacolata; Capasso, Mario; Iolascon, Achille

    2015-06-09

    Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease.

  7. Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

    PubMed

    Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

    2016-11-03

    Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H₂O₂-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H₂O₂-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

  8. Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1)

    PubMed Central

    Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

    2016-01-01

    Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage. PMID:27827892

  9. Oleanolic Acid Inhibits High Salt-Induced Exaggeration of Warburg-like Metabolism in Breast Cancer Cells.

    PubMed

    Amara, Suneetha; Zheng, Mu; Tiriveedhi, Venkataswarup

    2016-09-01

    Cancer cells have a proliferative advantage by utilizing intermediates of aerobic glycolysis (Warburg effect) for their macromolecule synthesis. Although the exact causes of this Warburg effect are unclear, high osmotic stress in solid tumor microenvironment is considered one of the important factors. Oleanolic acid (OA) is known to exert anti-inflammatory and anti-cancer effect. In our current studies, using breast cancer cell lines, we determined the protective role of OA in high salt-mediated osmotic stress-induced cancer growth. Hypertonic (0.16 M NaCl) culture conditions enhanced the cancer cell growth (26 %, p < 0.05) and aerobic glycolysis as marked by increased glucose consumption (34 %, p < 0.05) and lactate production (25 %, p < 0.05) over untreated cells. This effect was associated with increased expression and activity of key rate-limiting enzymes of aerobic glycolysis, namely hexokinase, pyruvate kinase type M2, and lactate dehydrogenase A. Interestingly, this high salt-mediated enhanced expression of aerobic glycolytic enzymes was efficiently reversed by OA along with the decreased cancer cell proliferation. In cancer cells, enhanced aerobic glycolysis is associated with the decreased mitochondrial activity and mitochondrial-associated caspase activity. As expected, high salt further inhibited the mitochondrial related cytochrome oxidase and caspase-3 activity. However, OA efficiently reversed the high salt-mediated inhibition of cytochrome oxidase, caspase activity, and pro-apoptotic Bax expression, thus suggesting that OA induced mitochondrial activity and enhanced apoptosis. Taken together, our data indicate that OA efficiently reverses the enhanced Warburg-like metabolism induced by high salt-mediated osmotic stress along with potential application of OA in anti-cancer therapy.

  10. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

    PubMed

    Aimola, Idowu A; Inuwa, Hajiya M; Nok, Andrew J; Mamman, Aisha I; Bieker, James J

    2016-04-05

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

  11. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

    PubMed Central

    Aimola, Idowu A.; Inuwa, Hajiya M.; Nok, Andrew J.; Mamman, Aisha I.; Bieker, James J.

    2017-01-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24 h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer PMID:26879870

  12. Gambogic acid potentiates the chemosensitivity of colorectal cancer cells to 5-fluorouracil by inhibiting proliferation and inducing apoptosis

    PubMed Central

    Wei, Jianchang; Yang, Ping; Li, Wanglin; He, Feng; Zeng, Shanqi; Zhang, Tong; Zhong, Junbin; Huang, Di; Chen, Zhuanpeng; Wang, Chengxing; Chen, Huacui; Hu, He; Cao, Jie

    2017-01-01

    Chemotherapy using 5-fluorouracil (5-FU) for colorectal cancer (CRC) has low specificity and response rates, leading to severe side effects. Gambogic acid (GA), a traditional Chinese medicine, has multi-targeted anticancer effects, including growth inhibition and apoptosis induction. However, it is unclear whether a combination of 5-FU and GA has synergistic anticancer effects in CRC cells. In this study, SW480 and HCT116 human CRC cells and human intestinal epithelial cells (IECs) were treated with different concentrations of 5-FU, GA or 5-FU+GA. A Cell Counting kit-8 assay was conducted to quantify cell proliferation. The combination index (CI) was calculated and the median-effect principle was applied to analyze the interaction between 5-FU and GA. Flow cytometry was used to determine the percentage of cells undergoing apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were applied to measure P53, survivin and thymidylate synthase (TS) mRNA and protein levels. It was found that 5-FU+GA more pronouncedly inhibited cell growth and induced apoptosis, compared with either monotherapy. CI values <1 indicated the synergistic effects of the drugs. 5-FU+GA further decreased P53, survivin and TS mRNA and protein levels in the two CRC cell lines compared with single drugs, whereas increased P53 protein levels were observed in HCT116 cells. Moreover, 5-FU+GA did not increase cytotoxicity to IECs. These results demonstrate that GA enhances the anticancer effects of 5-FU on CRC cells. Combined treatment with 5-FU and GA is effective and safe for CRC cells, and may become a promising chemotherapy treatment. PMID:28352348

  13. The pentacyclic triterpenoid, plectranthoic acid, a novel activator of AMPK induces apoptotic death in prostate cancer cells.

    PubMed

    Akhtar, Nosheen; Syed, Deeba N; Khan, Mohammad Imran; Adhami, Vaqar M; Mirza, Bushra; Mukhtar, Hasan

    2016-01-26

    Epidemiologic studies indicated that diabetics treated with metformin had a lower incidence of cancer than those taking other anti-diabetes drugs. This led to a surge in the efforts for identification of safer and more effective metformin mimetic compounds. The plant Ficus microcarpa is widely used for the treatment of type 2 diabetes in traditional medicine in South Asia. We obtained extracts from this plant and identified a small molecule, plectranthoic acid (PA), with potent 5'AMP-activated kinase (AMPK) activating properties far superior than metformin. AMPK is the central hub of metabolic regulation and a well-studied therapeutic target for metabolic syndrome, type-2 diabetes and cancer. We observed that treatment of prostate cancer (PCa) cells with PA inhibited proliferation and induced G0/G1 phase cell cycle arrest that was associated with up-regulation of cyclin kinase inhibitors p21/CIP1 and p27/KIP1. PA treatment suppressed mTOR/S6K signaling and induced apoptosis in PCa cells in an AMPK-dependent manner. Interestingly, PA-induced autophagy in PCa cells was found to be independent of AMPK activation. Combination studies of PA and metformin demonstrated that metformin had an inhibitory effect on PA-induced AMPK activation and suppressed PA-mediated apoptosis. Given the anti-proliferative role of PA in cancer and its potent anti-hyperglycemic activity, we suggest that PA should be explored further as a novel activator of AMPK for its ultimate use for the prevention of cancers and treatment of type 2 diabetes.

  14. The pentacyclic triterpenoid, plectranthoic acid, a novel activator of AMPK induces apoptotic death in prostate cancer cells

    PubMed Central

    Akhtar, Nosheen; Syed, Deeba N.; Khan, Mohammad Imran; Adhami, Vaqar M.; Mirza, Bushra; Mukhtar, Hasan

    2016-01-01

    Epidemiologic studies indicated that diabetics treated with metformin had a lower incidence of cancer than those taking other anti-diabetes drugs. This led to a surge in the efforts for identification of safer and more effective metformin mimetic compounds. The plant Ficus microcarpa is widely used for the treatment of type 2 diabetes in traditional medicine in South Asia. We obtained extracts from this plant and identified a small molecule, plectranthoic acid (PA), with potent 5′AMP-activated kinase (AMPK) activating properties far superior than metformin. AMPK is the central hub of metabolic regulation and a well-studied therapeutic target for metabolic syndrome, type-2 diabetes and cancer. We observed that treatment of prostate cancer (PCa) cells with PA inhibited proliferation and induced G0/G1 phase cell cycle arrest that was associated with up-regulation of cyclin kinase inhibitors p21/CIP1 and p27/KIP1. PA treatment suppressed mTOR/S6K signaling and induced apoptosis in PCa cells in an AMPK-dependent manner. Interestingly, PA-induced autophagy in PCa cells was found to be independent of AMPK activation. Combination studies of PA and metformin demonstrated that metformin had an inhibitory effect on PA-induced AMPK activation and suppressed PA-mediated apoptosis. Given the anti-proliferative role of PA in cancer and its potent anti-hyperglycemic activity, we suggest that PA should be explored further as a novel activator of AMPK for its ultimate use for the prevention of cancers and treatment of type 2 diabetes. PMID:26683363

  15. Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

    PubMed

    Im, Jintaek; Baik, Jung Eun; Kim, Kyoung Whun; Kang, Seok-Seong; Jeon, Jun Ho; Park, Ok-Jin; Kim, Hyun Young; Kum, Kee-Yeon; Yun, Cheol-Heui; Han, Seung Hyun

    2015-08-01

    Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

  16. MiR-652 inhibits acidic microenvironment-induced epithelial-mesenchymal transition of pancreatic cancer cells by targeting ZEB1

    PubMed Central

    Niu, Yi; Zhu, Shuai; Jin, Yan; Deng, Shijiang; Chen, Jingyuan; Liu, Yang; He, Chi; Yin, Tao; Yang, Zhiyong; Tao, Jing; Xiong, Jiongxin; Wu, Heshui; Wang, Chunyou; Zhao, Gang

    2015-01-01

    Recent evidences suggest that the acidic microenvironment might facilitate epithelial mesenchymal transition (EMT) of tumor cells, while the effects of acidity on EMT of pancreatic cancer (PC) remain undefined. The present study demonstrated that acidity suppressed miR-652 expression, which further promoted EMT process by absenting inhibition on the transcriptional factor ZEB1 expression. At first, we found that acidity remarkably enhanced invasion ability of PC cells accompanying with increased mesenchymal and decreased epithelial markers. Meanwhile, miRNAs-microarray showed that miR-652, the potential regulator of ZEB1, was distinctly decreased in acidity-treated PC cells. Furthermore, restoration of miR-652 reversed acidity-induced EMT by inhibiting ZEB1 expression, while miR-652 inhibitor induced EMT in normal PC cells through promoting ZEB1 expression. Nevertheless, knockdown of ZEB1 significantly suppressed acidity-induced EMT in PC cells, but ZEB1 overexpression rescued the EMT which was inhibited by miR-652 overexpression. The in vivo results showed that the tumor growth and liver metastasis were remarkably retarded by both miR-652 overexpression and ZEB1 knockdown. The clinical samples further revealed that miR-652 was decreased in PC tissues and antagonistically correlated with ZEB1 expression, associating with late tumor stage, lymphatic invasion and metastasis. In conclusion, our study indicated a novel acidity/miR-652/ZEB1/EMT axis in the tumorigenesis of PC. PMID:26498682

  17. Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca2+-dependent induction of oxidative stress

    PubMed Central

    Crnkovic, Slaven; Riederer, Monika; Lechleitner, Margarete; Hallström, Seth; Malli, Roland; Graier, Wolfgang F.; Lindenmann, Jörg; Popper, Helmut; Olschewski, Horst; Olschewski, Andrea; Frank, Saša

    2012-01-01

    Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca2+ release and entry of extracellular Ca2+. Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca2+ uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca2+-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs. PMID:22391221

  18. Ellagic acid inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and prevents atheroma formation in streptozotocin-induced diabetic rats.

    PubMed

    Rani, Uma P; Kesavan, Rushendhiran; Ganugula, Raghu; Avaneesh, T; Kumar, Uday P; Reddy, G Bhanuprakash; Dixit, Madhulika

    2013-11-01

    Plant-derived polyphenolic compounds have beneficial health effects. In the present study, we determined the ability of ellagic acid (EA) to prevent platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs). We also determined the ability of EA to prevent atherosclerosis in streptozotocin-induced diabetic rats. Proliferation of cells was measured via Alamar Blue assay and through propidium iodide-based cell cycle analysis in flow cytometer. Reactive oxygen species (ROS) were measured via 2',7'-dichlorofluorescin diacetate and Amplex red methods. Expression of proliferation markers and activation of kinases were assessed by immunoblot analysis. Cotreatment of primary cultures of RASMCs with 25 μmol/L of EA significantly reduced PDGF-BB (20 ng/ml)-induced proliferation by blocking S-phase entry. EA effectively blocked PDGF receptor-β (PDGFR-β) tyrosine phosphorylation, generation of intracellular ROS and downstream activation of extracellular signal-regulated kinase 1/2. It also blocked PDGF-BB-induced expression of cyclin D1. Computational molecular docking of EA with the PDGFR-β-PDGF-BB complex revealed two putative inhibitor binding sites which showed similar binding energies with the known PDGFR-β inhibitor AG1295. In diabetic rats, supplementation of diet with 2% EA significantly blocked diabetes-induced medial thickness, and lipid and collagen deposition in the arch of aorta. These were assessed through haematoxylin and eosin, Oil Red O and Masson's trichome staining, respectively. EA treatment also blocked cyclin D1 expression in medial smooth muscle cells in experimental animals. Thus, EA is effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells.

  19. A sequential treatment regimen with melatonin and all-trans retinoic acid induces apoptosis in MCF-7 tumour cells.

    PubMed Central

    Eck, K. M.; Yuan, L.; Duffy, L.; Ram, P. T.; Ayettey, S.; Chen, I.; Cohn, C. S.; Reed, J. C.; Hill, S. M.

    1998-01-01

    Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken

  20. New betulinic acid derivatives induce potent and selective antiproliferative activity through cell cycle arrest at the S phase and caspase dependent apoptosis in human cancer cells.

    PubMed

    Santos, Rita C; Salvador, Jorge A R; Cortés, Roldán; Pachón, Gisela; Marín, Silvia; Cascante, Marta

    2011-06-01

    New semisynthetic derivatives of betulinic acid (BA) RS01, RS02 and RS03 with 18-45 times improved cytotoxic activity against HepG2 cells, were tested for their ability to induce apoptosis and cell cycle arrest in HepG2, HeLa and Jurkat cells. All the compounds induced significant increase in the population at the S phase more effectively than BA. RS01, RS02 and RS03 were also found to be potent inducers of apoptosis with RS01 being markedly more potent than BA, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell cycle arrest. The mechanism of apoptosis induction has been studied in HepG2 cells and found to be mediated by activation of the postmitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8. These facts were corroborated by detection of mitochondrial cytochrome c release and DNA fragmentation. Because RS01, RS02 and RS03 exhibited significant improved antitumor activity with respect to BA, they may be promising new agents for the treatment of cancer. In particular, RS01 is the most promising compound with an IC(50) value 45 times lower than BA on HepG2 cells and 61 times lower than the one found for the non-tumoral Chang liver cells.

  1. The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells.

    PubMed

    Goldman, Aaron; Chen, HwuDauRw; Khan, Mohammad R; Roesly, Heather; Hill, Kimberly A; Shahidullah, Mohammad; Mandal, Amritlal; Delamere, Nicholas A; Dvorak, Katerina

    2011-01-01

    Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.

  2. Knockdown of XAB2 enhances all-trans retinoic acid-induced cellular differentiation in all-trans retinoic acid-sensitive and -resistant cancer cells.

    PubMed

    Ohnuma-Ishikawa, Kumiko; Morio, Tomohiro; Yamada, Takayuki; Sugawara, Yuji; Ono, Makoto; Nagasawa, Masayuki; Yasuda, Akio; Morimoto, Chikao; Ohnuma, Kei; Dang, Nam H; Hosoi, Hajime; Verdin, Eric; Mizutani, Shuki

    2007-02-01

    Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.

  3. 5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines.

    PubMed

    Sharma, Sulbha; Jajoo, Anjana; Dube, Alok

    2007-09-25

    Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.

  4. Combined treatment with the Cox-2 inhibitor niflumic acid and PPARγ ligand ciglitazone induces ER stress/caspase-8-mediated apoptosis in human lung cancer cells.

    PubMed

    Kim, Byeong Mo; Maeng, Kyungah; Lee, Kee-Ho; Hong, Sung Hee

    2011-01-28

    The present study was performed to investigate the possible combined use of the Cox-2 inhibitor niflumic acid and the PPARγ ligand ciglitazone and to elucidate the mechanisms underlying enhanced apoptosis by this combination treatment in human lung cancer cells. Combined niflumic acid-ciglitazone treatment synergistically induced apoptotic cell death, activated caspase-9, caspase-3, and induced caspase-3-mediated PARP cleavage. The combination treatment also triggered apoptosis through caspase-8/Bid/Bax activation, and the inhibition of caspase-8 suppressed caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and concomitant apoptosis. In addition, combined niflumic acid-ciglitazone treatment significantly induced ER stress responses, and suppression of CHOP expression significantly attenuated the combined niflumic acid-ciglitazone treatment-induced activation of caspase-8 and caspase-3, and the subsequent apoptotic cell death, indicating a role of ER stress in caspase-8 activation and apoptosis. Interestingly, the pro-apoptotic effects of combined niflumic acid-ciglitazone treatment were realized through Cox-2- and PPARγ-independent mechanisms. Taken together, these results suggest that sequential ER stress and caspase-8 activation are critical in combined niflumic acid-ciglitazone treatment-induced apoptosis in human lung cancer cells.

  5. Farnesoid X receptor signal is involved in deoxycholic acid-induced intestinal metaplasia of normal human gastric epithelial cells.

    PubMed

    Li, Shu; Chen, Xin; Zhou, Lu; Wang, Bang-Mao

    2015-11-01

    The farnesoid X receptor (FXR) signaling pathway is known to be involved in the metabolism of bile acid, glucose and lipid. In the present study, we demonstrated that 400 µmol/l deoxycholic acid (DCA) stimulation promotes the proliferation of normal human gastric epithelial cells (GES-1). In addition, DCA activated FXR and increased the expression of intestinal metaplasia genes, including caudal-related homeobox transcription factor 2 (Cdx2) and mucin 2 (MUC2). The treatment of FXR agonist GW4064/antagonist guggulsterone (Gug.) significantly increased/decreased the expression levels of FXR, Cdx2 and MUC2 protein in DCA-induced GES-1 cells. GW4064/Gug. also enhanced/reduced the nuclear factor-κB (NF-κB) activity and binding of the Cdx2 promoter region and NF-κB, the most common subunit p50 protein. Taken together, the results indicated that DCA is capable of modulating the expression of Cdx2 and the downstream MUC2 via the nuclear receptor FXR-NF-κB activity in normal gastric epithelial cells. FXR signaling pathway may therefore be involved in the intestinal metaplasia of human gastric mucosa.

  6. Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

    PubMed

    Jung, Kwangseon; Cho, Jae Youl; Soh, Young-Jin; Lee, Jienny; Shin, Seoung Woo; Jang, Sunghee; Jung, Eunsun; Kim, Min Hee; Lee, Jongsung

    2015-01-01

    Ultraviolet A (UVA) irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate effects of aspartic acid on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Furthermore, we elucidated the UVA-antagonizing mechanisms of aspartic acid. The results of this study showed that aspartic acid attenuated the UVA-induced reduction of the proliferative potential and stemness of hAMSCs, as evidenced by increased proliferative activity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and upregulation of stemness-related genes OCT4, NANOG, and SOX2 in response to the aspartic acid treatment. UVA-induced reduction in the mRNA level of hypoxia-inducible factor (HIF)-1α was also significantly recovered by aspartic acid. In addition, the antagonizing effects of aspartic acid against the UVA effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show that aspartic acid improves reduced stemness of hAMSCs induced by UVA and its effects are mediated by upregulation of HIF-1α via the inhibition of PGE2-cAMP signaling. In addition, aspartic acid may be used as an antagonizing agent to mitigate the effects of UVA.

  7. Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells

    PubMed Central

    Lee, Jienny; Shin, Seoung Woo; Jang, Sunghee; Jung, Eunsun; Kim, Min Hee; Lee, Jongsung

    2015-01-01

    Ultraviolet A (UVA) irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate effects of aspartic acid on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Furthermore, we elucidated the UVA-antagonizing mechanisms of aspartic acid. The results of this study showed that aspartic acid attenuated the UVA-induced reduction of the proliferative potential and stemness of hAMSCs, as evidenced by increased proliferative activity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and upregulation of stemness-related genes OCT4, NANOG, and SOX2 in response to the aspartic acid treatment. UVA-induced reduction in the mRNA level of hypoxia-inducible factor (HIF)-1α was also significantly recovered by aspartic acid. In addition, the antagonizing effects of aspartic acid against the UVA effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show that aspartic acid improves reduced stemness of hAMSCs induced by UVA and its effects are mediated by upregulation of HIF-1α via the inhibition of PGE2-cAMP signaling. In addition, aspartic acid may be used as an antagonizing agent to mitigate the effects of UVA. PMID:25909857

  8. Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Clohisy, J. C.; Connolly, T. J.; Bergman, K. D.; Quinn, C. O.; Partridge, N. C.

    1994-01-01

    Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

  9. Pancreatic cancer-specific cell death induced in vivo by cytoplasmic-delivered polyinosine-polycytidylic acid

    PubMed Central

    Bhoopathi, Praveen; Quinn, Bridget A.; Gui, Qin; Shen, Xue-Ning; Grossman, Steven R.; Das, Swadesh K.; Sarkar, Devanand; Fisher, Paul B.; Emdad, Luni

    2014-01-01

    Polyinosine-polycytidylic acid (pIC) is a synthetic dsRNA that acts as an immune agonist of TLR3 and RLR to activate dendritic and NK cells that can kill tumor cells. pIC can also trigger apoptosis in pancreatic ductal adenocarcinoma cells but its mechanism of action is obscure. In this study, we investigated the potential therapeutic activity of a formulation of pIC with polyethylenimine ([pIC]PEI) in PDAC and investigated its mechanism of action. [pIC]PEI stimulated apoptosis in PDAC cells without affecting normal pancreatic epithelial cells. Mechanistically, [pIC]PEI repressed XIAP and survivin expression and activated an immune response by inducing MDA-5, RIG-I and NOXA. Phosphorylation of AKT was inhibited by [pIC]PEI in PDAC and this event was critical for stimulating apoptosis through XIAP and survivin degradation. In vivo administration of [pIC]PEI inhibited tumor growth via AKT-mediated XIAP degradation in both subcutaneous and quasi-orthotopic-models of PDAC. Taken together, these results offer a preclinical proof-of-concept for the evaluation of [pIC]PEI as an immunochemotherapy to treat pancreatic cancer. PMID:25205107

  10. [Expression change of IL-3 receptor system in all-trans retinoic acid induced differentiation of NB4 cells].

    PubMed

    Wu, Yong; Yang, Jing-Hui; Li, Xian-Fang; Liao, Xiao-Ying; Huang, Hui-Fang; Chen, Yuan-Zhong

    2010-12-01

    Interleukin-3 receptor (IL-3R) is a heterodimeric membrane receptor. The α subunit is essential for ligand binding and confers ligand specificity to the receptor. The common beta chain (βc) subunit, which is shared by the granulocyte macrophage-colony stimulating factor (GM-CSF), IL-3 and IL-5 receptors, is required for high-affinity ligand binding and signal transduction, mediating growth and survival of hematopoietic progenitor cells and the production and activation of mature hematopoietic cells. In order to investigate the role of IL-3 receptor system (IL-3Rα, GM-CSFRα and hβc) in myeloid differentiation, the expression level of IL-3 receptor system gene in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by quantitative real time RT-PCR. At the same time, DNA sequence change was analyzed by cDNA sequencing. The results showed that the expression level of IL-3Rα mRNA was obviously down-regulated in NB4 cells treated with ATRA for 24 hours, but during differentiation of ATRA induced NB4 cells, the expression level of IL-3Rα mRNA was gradually restored, while the expression levels of GM-CSFRα mRNA and hβc mRNA were gradually up-regulated. The sequence of IL-3Rα and GM-CSFRα gene did not change before and after NB4 cells differentiation, but the sequence of hβc gene changed when NB4 cells were treated with ATRA, the expression of hβc mRNA sequence before NB4 cell differentiation taken truncated mutation as dominant, as regards expression of hβc mRNA sequence after NB4 cell differentiation, the truncated mutation of hβc mRNA had restored to wild type. It is concluded that the IL-3 receptor abnormality exists in NB4 cells, over expression of IL-3Rα and truncated mutation of hβc may be involved in proliferation and differentiation block in NB4 cells.

  11. Chloride-inducible transient apoplastic alkalinizations induce stomata closure by controlling abscisic acid distribution between leaf apoplast and guard cells in salt-stressed Vicia faba.

    PubMed

    Geilfus, Christoph-Martin; Mithöfer, Axel; Ludwig-Müller, Jutta; Zörb, Christian; Muehling, Karl H

    2015-11-01

    Chloride stress causes the leaf apoplast transiently to alkalize, an event that is presumed to contribute to the ability of plants to adapt to saline conditions. However, the initiation of coordinated processes downstream of the alkalinization is unknown. We hypothesize that chloride-inducible pH dynamics are a key chemical feature modulating the compartmental distribution of abscisic acid (ABA) and, as a consequence, affecting stomata aperture. Apoplastic pH and stomata aperture dynamics in intact Vicia faba leaves were monitored by microscopy-based ratio imaging and porometric measurements of stomatal conductance. ABA concentrations in leaf apoplast and guard cells were compared with pH dynamics by gas-chromatography-mass-spectrometry (GC-MS) and liquid-chromatography-tandem-mass spectrometry (LC-MS/MS). Results demonstrate that, upon chloride addition to roots, an alkalizing factor that initiates the pH dynamic propagates from root to leaf in a way similar to xylem-distributed water. In leaves, it induces a systemic transient apoplastic alkalinization that causes apoplastic ABA concentration to increase, followed by an elevation of endogenous guard cell ABA. We conclude that the transient alkalinization, which is a remote effect of chloride stress, modulates the compartmental distribution of ABA between the leaf apoplast and the guard cells and, in this way, is instrumental in inducing stomata closure during the beginning of salinity.

  12. Photodynamic therapy with 5-aminoolevulinic acid-induced porphyrins and DMSO/EDTA for basal cell carcinoma

    NASA Astrophysics Data System (ADS)

    Warloe, Trond; Peng, Qian; Heyerdahl, Helen; Moan, Johan; Steen, Harald B.; Giercksky, Karl-Erik

    1995-03-01

    Seven hundred sixty three basal cell carcinomas (BCCs) in 122 patients were treated by photodynamic therapy by 5-aminolevulinic acid (ALA) in cream topically applied, either alone, in combination with dimethyl sulphoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA), or with DMSO as a pretreatment. After 3 hours cream exposure 40 - 200 Joules/cm2 of 630 nm laser light was given. Fluorescence imaging of biopsies showed highly improved ALA penetration depth and doubled ALA-induced porphyrin production using DMSO/EDTA. Treatment response was recorded after 3 months. After a single treatment 90% of 393 superficial lesions responded completely, independent of using DMSO/EDTA. In 363 nodulo-ulcerative lesions the complete response rate increased from 67% to above 90% with DMSO/EDTA for lesions less than 2 mm thickness and from 34% to about 50% for lesions thicker than 2 mm. Recurrence rate observed during a follow-up period longer than 12 months was 2 - 5%. PDT of superficial thin BCCs with ALA-induced porphyrins and DMSO/EDTA equals surgery and radiotherapy with respect to cure rate and recurrence. Cosmetic results of ALA-based PDT seemed to be better than those after other therapies. In patients with the nevoid BCC syndrome the complete response rate after PDT was far lower.

  13. Fenofibrate, a PPARα agonist, protect proximal tubular cells from albumin-bound fatty acids induced apoptosis via the activation of NF-kB.

    PubMed

    Zuo, Nan; Zheng, Xiaoyu; Liu, Hanzhe; Ma, Xiaoli

    2015-01-01

    Albumin-bound fatty acids is the main cause of renal damage, PPARα is responsible in the metabolism of fatty acids. Previous study found that PPARα played a protective role in fatty acids overload associated tubular injury. The aim of the present study is to investigate whether fenofibrate, a PPARα ligands, could contribute to the renoprotective action in fatty acids overload proximal tubule epithelial cells. We observed in HK-2 cells that fenofibrate significantly inhibited fatty acids bound albumin (FA-BSA) induced up-regulation of MCP-1 and IL-8. Treatment with fenofibrate attenuated renal oxidative stress induced by FA-BSA as evidenced by decreased MDA level, increased SOD activity and catalase, GPx-1 expression. FA-BSA induced apoptosis of HK-2 cells were also obviously prevented by fenofibrate. Furthermore, fenofibrate significantly increased the expression of PPARα mRNA and protein in FA-BSA treated cells. Finally, the activation of NF-kB induced by FA-BSA was markedly suppressed by fenofibrate. Taken together, our study describes a renoprotective role of fenofibrate in fatty acids associated tubular toxicity, and the transcriptional activation of PPARα and suppression of NF-kB were at least partially involved.

  14. Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

    PubMed

    Hsu, S L; Cheng, C C; Shi, Y R; Chiang, C W

    2001-06-26

    Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

  15. Biocontrol agents-mediated suppression of oxalic acid induced cell death during Sclerotinia sclerotiorum-pea interaction.

    PubMed

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Sarma, Birinchi Kumar; Singh, Harikesh Bahadur

    2015-05-01

    Oxalic acid (OA) is an important pathogenic factor during early Sclerotinia sclerotiorum-host interaction and might work by reducing hydrogen peroxide production (H2 O2 ). In the present investigation, oxalic acid-induced cell death in pea was studied. Pea plants treated with biocontrol agents (BCAs) viz., Pseudomonas aeruginosa PJHU15, Bacillus subtilis BHHU100, and Trichoderma harzianum TNHU27 either singly and/or in consortium acted on S. sclerotiorum indirectly by enabling plants to inhibit the OA-mediated suppression of oxidative burst via induction of H2 O2 . Our results showed that BCA treated plants upon treatment with culture filtrate of the pathogen, conferred the resistance via. significantly decreasing relative cell death of pea against S. sclerotiorum compared to control plants without BCA treatment but treated with the culture filtrate of the pathogen. The results obtained from the present study indicate that the microbes especially in consortia play significant role in protection against S. sclerotiorum by modulating oxidative burst and partially enhancing tolerance by increasing the H2 O2 generation, which is otherwise suppressed by OA produced by the pathogen.

  16. Cichoric Acid Reverses Insulin Resistance and Suppresses Inflammatory Responses in the Glucosamine-Induced HepG2 Cells.

    PubMed

    Zhu, Di; Wang, Yutang; Du, Qingwei; Liu, Zhigang; Liu, Xuebo

    2015-12-30

    Cichoric acid, a caffeic acid derivative found in Echinacea purpurea, basil, and chicory, has been reported to have bioactive effects, such as anti-inflammatory, antioxidant, and preventing insulin resistance. In this study, to explore the effects of CA on regulating insulin resistance and chronic inflammatory responses, the insulin resistance model was constructed by glucosamine in HepG2 cells. CA stimulated glucosamine-mediated glucose uptake by stimulating translocation of the glucose transporter 2. Moreover, the production of reactive oxygen, the expression of COX-2 and iNOS, and the mRNA levels of TNF-α and IL-6 were attenuated. Furthermore, CA was verified to promote glucosamine-mediated glucose uptake and inhibited inflammation through PI3K/Akt, NF-κB, and MAPK signaling pathways in HepG2 cells. These results implied that CA could increase glucose uptake, improve insulin resistance, and attenuate glucosamine-induced inflammation, suggesting that CA is a potential natural nutraceutical with antidiabetic properties and anti-inflammatory effects.

  17. Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

    PubMed Central

    2005-01-01

    We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal. PMID:15673285

  18. EPA, an omega-3 fatty acid, induces apoptosis in human pancreatic cancer cells: role of ROS accumulation, caspase-8 activation, and autophagy induction.

    PubMed

    Fukui, Masayuki; Kang, Ki Sung; Okada, Kazushi; Zhu, Bao Ting

    2013-01-01

    In a recent study, we showed that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two common omega-3 fatty acids, can cause ROS accumulation and subsequently induce caspase-8-dependent apoptosis in human breast cancer cells (Kang et al. [2010], PLoS ONE 5: e10296). In this study, we showed that the pancreas has a unique ability to accumulate EPA at a level markedly higher than several other tissues analyzed. Based on this finding, we sought to further investigate the anticancer actions of EPA and its analog DHA in human pancreatic cancer cells using both in vitro and in vivo models. EPA and DHA were found to induce ROS accumulation and caspase-8-dependent cell death in human pancreatic cancer cells (MIA-PaCa-2 and Capan-2) in vitro. Feeding animals with a diet supplemented with 5% fish oil, which contains high levels of EPA and DHA, also strongly suppresses the growth of MIA-PaCa-2 human pancreatic cancer xenografts in athymic nude mice, by inducing oxidative stress and cell death. In addition, we showed that EPA can concomitantly induce autophagy in these cancer cells, and the induction of autophagy diminishes its ability to induce apoptotic cell death. It is therefore suggested that combination of EPA with an autophagy inhibitor may be a useful strategy in increasing the therapeutic effectiveness in pancreatic cancer.

  19. HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

    PubMed

    Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

    2015-01-01

    Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury.

  20. All-trans retinoic acid and genistein induce cell apoptosis in OVCAR-3 cells by increasing the P14 tumor suppressor gene

    PubMed Central

    Dastjerdi, Mehdi Nikbakht; Zamani, Saeed; Mardani, Mohammad; Beni, Batool Hashemi

    2016-01-01

    In this study, we evaluated the effects of all-trans retinoic acid (ATRA) alone or in combination with genistein (GEN) in p14 tumor suppressor gene and subsequent apoptosis of human ovarian carcinoma cells (OVCAR-3). The cells were treated with ATRA or GEN at concentrations of 50 and 25 μM respectively, either alone or in combination for 24 and 48 h. The cell viability was evaluated using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The percentage of cell apoptosis was determined using flow cytometry and p14 gene expression was measured using real time PCR. The MTT results showed that in both ATRA and GEN treated groups, the cell viabilityviability in group treated for 48 h was significantly lower than group treated for 24 h. The flow cytometry results showed that the percentage of apoptotic cells in groups that treated with ATRA and GEN in combination for 24 h and 48 h was significantly more than all other tested groups. The real time results showed that the mRNA level of p14 in cells treated with both drugs for 48 h was significantly higher than all other groups. In conclusion, we confirm that GEN in combination with ATRA is an effective strategy to up regulate the p14 tumor suppressor gene and induce cell apoptosis in OVCAR-3 cell line. PMID:28003845

  1. Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

    PubMed

    Sutton-McDowall, Melanie L; Wu, Linda L Y; Purdey, Malcolm; Abell, Andrew D; Goldys, Ewa M; MacMillan, Keith L; Thompson, Jeremy G; Robker, Rebecca L

    2016-01-01

    Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

  2. Effect of royal jelly on experimental colitis induced by acetic acid and alteration of mast cell distribution in the colon of rats

    PubMed Central

    Karaca, T.; Bayiroglu, F.; Yoruk, M.; Kaya, M.S.; Uslu, S.; Comba, B.; Mis, L.

    2010-01-01

    This study investigated the effects of royal jelly (RJ) on acetic acid-induced colitis in rats. Twenty adult female Wistar albino rats were divided into four treatment groups of 5 animals each, including a control group (Group I); Group II was treated orally with RJ (150 mg kg−1 body weight); Group III had acetic acid-induced colitis; and Group IV had acetic acid-induced colitis treated orally with RJ (150 mg kg−1 body weight) for 4 weeks. Colitis was induced by intracolonic instillation of 4% acetic acid; the control group received physiological saline (10 mL kg−1). Colon samples were obtained under deep anaesthesia from animals in all groups. Tissues were fixed in 10% formalin neutral buffer solution for 24 h and embedded in paraffin. Six-micrometre-thick sections were stained with Mallory’s triple stain and toluidine blue in 1% aqueous solution at pH 1.0 for 5 min (for Mast Cells). RJ was shown to protect the colonic mucosa against the injurious effect of acetic acid. Colitis (colonic damage) was confirmed histomorphometrically as significant increases in the number of mast cells (MC) and colonic erosions in rats with acetic acid-induced colitis. The RJ treatment significantly decreased the number of MC and reduced the area of colonic erosion in the colon of RJ-treated rats compared with rats with untreated colitis. The results suggest that oral treatment with RJ could be used to treat colitis. PMID:21263740

  3. Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation.

    PubMed

    Chien, Chih-Chiang; Wu, Ming-Shun; Shen, Shing-Chuan; Yang, Liang-Yo; Wu, Wen-Shin; Chen, Yen-Chou

    2013-04-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.

  4. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    PubMed

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  5. Distribution of interstitial cells of Cajal in the bladders of fetal rats with retinoic acid induced myelomeningocele

    PubMed Central

    Tekin, Ali; Karakuş, Osman Zeki; Hakgüder, Gülce; Ateş, Oğuz; Özer, Erdener; Olguner, Mustafa; Akgür, Feza Miraç

    2016-01-01

    Objective Myelomeningocele (MMC) is one of the most common reason of neurogenic bladder dysfunction in children. Although neurogenic bladder dysfunction occurrence is related with bladder innervation, also there are some changes seen in the smooth muscle and neural cells of the bladder. Interstitial cells of Cajal (ICC) are the pacemaker cells found in organs with peristaltic activity. Although it has been shown that ICC are diminished in the rat urinary bladder with traumatic spinal cord injury, there is no data about ICC in fetal rat bladders with MMC. This study has been conducted to investigate the ICC in the bladders of fetal rats with retinoic acid induced MMC. Materials and methods Time dated pregnant Wistar albino rats were divided into 3 groups. In MMC group, dams were fed with gavage solution containing 60 mg/kg all-trans retinoic acid dissolved in olive oil on 10. embryologic day. Sham group animals were fed only olive oil. Control group dams were fed with standard rat chow. Fetuses were delivered by cesarean section and harvested on 22. embryologic day. MMC was identified by observing MMC sacs at the back of the fetuses. Distribution of ICCs were evaluated using immunohistochemical staining. Results ICCs were found in all groups, which have the same morphological features that had been described earlier in the gastrointestinal tract and the bladder. The density of the ICC in the MMC group was found to be significantly decreased when compared with the control and the sham groups (p<0.05). Conclusion The density of the ICC in the urinary bladder decreased in the neurogenic bladder developed in MMC. PMID:27909623

  6. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis, down-regulates the CXCR4 chemokine receptor and impairs migration of chronic lymphocytic leukemia cells

    PubMed Central

    Stamatopoulos, Basile; Meuleman, Nathalie; De Bruyn, Cécile; Delforge, Alain; Bron, Dominique; Lagneaux, Laurence

    2010-01-01

    Background Chronic lymphocytic leukemia is a neoplastic disorder that arises largely as a result of defective apoptosis leading to chemoresistance. Stromal cell-derived factor-1 and its receptor, CXCR4, have been shown to play an important role in chronic lymphocytic leukemia cell trafficking and survival. Design and Methods Since histone acetylation is involved in the modulation of gene expression, we evaluated the effects of suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, on chronic lymphocytic leukemia cells and in particular on cell survival, CXCR4 expression, migration, and drug sensitization. Results Here, we showed that treatment with suberoylanilide hydroxamic acid (20 μM) for 48 hours induced a decrease in chronic lymphocytic leukemia cell viability via apoptosis (n=20, P=0.0032). Using specific caspase inhibitors, we demonstrated the participation of caspases-3, -6 and -8, suggesting an activation of the extrinsic pathway. Additionally, suberoylanilide hydroxamic acid significantly decreased CXCR4 mRNA (n=10, P=0.0010) and protein expression (n=40, P<0.0001). As a result, chronic lymphocytic leukemia cell migration in response to stromal cell-derived factor-1 (n=23, P<0.0001) or through bone marrow stromal cells was dramatically impaired. Consequently, suberoylanilide hydroxamic acid reduced the protective effect of the microenvironment and thus sensitized chronic lymphocytic leukemia cells to chemotherapy such as fludarabine. Conclusions In conclusion, suberoylanilide hydroxamic acid induces apoptosis in chronic lymphocytic leukemia cells via the extrinsic pathway and down-regulates CXCR4 expression leading to decreased cell migration. Suberoylanilide hydroxamic acid in combination with other drugs represents a promising therapeutic approach to inhibiting migration, chronic lymphocytic leukemia cell survival and potentially overcoming drug resistance. PMID:20145270

  7. RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae.

    PubMed

    Dong, Yachen; Hu, Jingjin; Fan, Linlin; Chen, Qihe

    2017-02-17

    As a typical harmful inhibitor in cellulosic hydrolyzates, acetic acid not only hinders bioethanol production, but also induces cell death in Saccharomyces cerevisiae. Herein, we conducted both transcriptomic and metabolomic analyses to investigate the global responses under acetic acid stress at different stages. There were 295 up-regulated and 427 down-regulated genes identified at more than two time points during acetic acid treatment (150 mM, pH 3.0). These differentially expressed genes (DEGs) were mainly involved in intracellular homeostasis, central metabolic pathway, transcription regulation, protein folding and stabilization, ubiquitin-dependent protein catabolic process, vesicle-mediated transport, protein synthesis, MAPK signaling pathways, cell cycle, programmed cell death, etc. The interaction network of all identified DEGs was constructed to speculate the potential regulatory genes and dominant pathways in response to acetic acid. The transcriptional changes were confirmed by metabolic profiles and phenotypic analysis. Acetic acid resulted in severe acidification in both cytosol and mitochondria, which was different from the effect of extracellular pH. Additionally, the imbalance of intracellular acetylation was shown to aggravate cell death under this stress. Overall, this work provides a novel and comprehensive understanding of stress responses and programmed cell death induced by acetic acid in yeast.

  8. RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae

    PubMed Central

    Dong, Yachen; Hu, Jingjin; Fan, Linlin; Chen, Qihe

    2017-01-01

    As a typical harmful inhibitor in cellulosic hydrolyzates, acetic acid not only hinders bioethanol production, but also induces cell death in Saccharomyces cerevisiae. Herein, we conducted both transcriptomic and metabolomic analyses to investigate the global responses under acetic acid stress at different stages. There were 295 up-regulated and 427 down-regulated genes identified at more than two time points during acetic acid treatment (150 mM, pH 3.0). These differentially expressed genes (DEGs) were mainly involved in intracellular homeostasis, central metabolic pathway, transcription regulation, protein folding and stabilization, ubiquitin-dependent protein catabolic process, vesicle-mediated transport, protein synthesis, MAPK signaling pathways, cell cycle, programmed cell death, etc. The interaction network of all identified DEGs was constructed to speculate the potential regulatory genes and dominant pathways in response to acetic acid. The transcriptional changes were confirmed by metabolic profiles and phenotypic analysis. Acetic acid resulted in severe acidification in both cytosol and mitochondria, which was different from the effect of extracellular pH. Additionally, the imbalance of intracellular acetylation was shown to aggravate cell death under this stress. Overall, this work provides a novel and comprehensive understanding of stress responses and programmed cell death induced by acetic acid in yeast. PMID:28209995

  9. Lipoic acid inhibits caspase-dependent and -independent cell death pathways and is neuroprotective against hippocampal damage after pilocarpine-induced seizures.

    PubMed

    dos Santos, Pauline Sousa; Feitosa, Chistiane Mendes; Saldanha, Gláucio Barros; Tomé, Adriana da Rocha; Feng, Dejiang; de Freitas, Rivelilson Mendes

    2011-01-01

    Alpha-lipoic acid has some neuroprotective properties, but this action has not been investigated in models of epilepsy. The aim of the present study was to investigate the protective efficacy of α-lipoic acid (lipoic acid) against pilocarpine-induced cell death through the caspase-dependent or -independent mitochondrial apoptotic pathways. Wistar rats were injected intraperitoneally with 0.9% saline (control group), pilocarpine (400 mg/kg, pilocarpine group) alone, or α-lipoic acid (20 mg/kg) in association with pilocarpine (400 mg/kg) 30 min before administration of α-lipoic acid. After the treatments all groups were observed for 24 h. Cell death was reduced in lipoic acid-treated rats. Cytosolic translocation of cytochrome c and subsequent activation of caspase-3 were reduced by lipoic acid treatment. AIF nuclear translocation and subsequent large-scale DNA fragmentation were also decreased in lipoic acid-treated rats. Our study suggests that lipoic acid inhibits both caspase-dependent and -independent apoptotic pathways and may be neuroprotective against hippocampal damage during pilocarpine-induced seizures.

  10. Antiapoptotic role of S-adenosyl-l-methionine against hydrochloric acid induced cell death in Saccharomyces cerevisiae.

    PubMed

    Malakar, Dipankar; Dey, Anindya; Basu, Arghya; Ghosh, Anil K

    2008-01-01

    Exposure of stationary phase cells of Saccharomyces cerevisiae to 10 mM HCl (pH approximately 2) resulted in cell death as a function of time (up to 6 h) with most (about 40%-65%) of the cells showing apoptotic features including chromatin condensation along the nuclear envelope, exposure of phosphatidylserine on the outer leaflet of cytoplasmic membrane, and DNA fragmentation. During the first 2 h of acid exposure there was an increase in reactive oxygen species (ROS) level inside cells, with subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents culminating in loss of mitochondrial membrane potential (DeltaPsi(m)). An initial (1 h) event of mitochondrial hyper-polarization with subsequent elevation of ROS level of the acid treated cells was also observed. S-adenosyl-l-methionine (AdoMet; 1 mM) treatment increased the cell survival of the acid stressed cells. It partially scavenged the increased intracellular ROS level by supplementing glutathione through the transsulfuration pathway. It also inhibited acid mediated lipid peroxidation, partially recovered acid evoked loss of DeltaPsi(m) and protected the cells from apoptotic cell death. S-adenosyl di-aldehyde, an indirect inhibitor of the AdoMet metabolic pathway, increased mortality of the acid treated cells. Incubation of acid stressed cells with the antioxidant, N-acetyl-cysteine (1 mM), decreased the cellular mortality, but the same concentration of AdoMet offered more protection by scavenging the free radicals. The ability of AdoMet to scavenge ROS mediated apoptosis may be an important function of this molecule in responding to cellular stress. The study could open a new avenue for detailed investigation on the curative potential of AdoMet against gastric ulcer.

  11. Interleukin-8 (IL-8) over-production and autocrine cell activation are key factors in monomethylarsonous acid [MMA(III)]-induced malignant transformation of urothelial cells.

    PubMed

    Escudero-Lourdes, C; Wu, T; Camarillo, J M; Gandolfi, A J

    2012-01-01

    The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation.

  12. Treadmill exercise ameliorates motor dysfunction through inhibition of Purkinje cell loss in cerebellum of valproic acid-induced autistic rats

    PubMed Central

    Cho, Han-Sam; Kim, Tae-Woon; Ji, Eun-Sang; Park, Hye-Sang; Shin, Mal-Soon; Baek, Seung-Soo

    2016-01-01

    Autism is a complex developmental disorder with impairments in social interaction, communication, repetitive behavior and motor skills. Exercise enhances cognitive function, ameliorates motor dysfunction, and provides protective profits against neurodegeneration. In the present study, we evaluated the effect of treadmill exercise on the motor coordination and Purkinje cell loss in relation with reactive astrocytes and microglial activation in the cerebellum using valproic acid (VPA)-induced autism rat model. On the 12th day of pregnancy, the pregnant rats in the VPA-exposed group received intraperitoneal injections of 600-mg/kg VPA. After birth, the rat pups were divided into four groups: the control group, the exercise group, the VPA-treated group, the VPA-treated and exercise group. The rat pups in the exercise groups were forced to run on a treadmill for 30 min once a day, 5 times a week for 4 weeks. In the present results, motor balance and coordination was disturbed by induction of autism, in contrast, treadmill exercise alleviated motor dysfunction in the autistic rats. Purkinje cell loss, reactive astrocytes, and microglial activation were occurred by induction of autism, in contrast, treadmill exercise enhanced survival rate of Purkinje neurons through inhibition of reactive astrocytes and microglia in the autistic rats. The present study showed that exercise may provide a potential therapeutic strategy for the alleviation of motor dysfunction in autistic patients. PMID:27656625

  13. Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells

    PubMed Central

    Angrisano, T.; Sacchetti, S.; Natale, F.; Cerrato, A.; Pero, R.; Keller, S.; Peluso, S.; Perillo, B.; Avvedimento, V. E.; Fusco, A.; Bruni, C. B.; Lembo, F.; Santoro, M.; Chiariotti, L.

    2011-01-01

    Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci. PMID:20952403

  14. Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.

    PubMed

    Babot, Zoila; Cristòfol, Rosa; Suñol, Cristina

    2005-01-01

    Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl

  15. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells.

    PubMed

    Kumar, Devbrat; Basu, Soumya; Parija, Lucy; Rout, Deeptimayee; Manna, Sanjeet; Dandapat, Jagneshwar; Debata, Priya Ranjan

    2016-07-01

    Cervical cancer and precancerous lesions of the cervix continue to be a global health issue, and the medication for the treatment for chronic HPV infection so far has not been effective. Potential anticancer and anti HPV activities of two known phytochemicals, Curcumin and Ellagic acid were evaluated in HeLa cervical cancer cells. Curcumin is a natural compound found in the root of Curcuma longa plant and Ellagic acid a polyphenol found in fruits of strawberries, raspberries and walnuts. The combination of Curcumin and Ellagic acid at various concentrations showed better anticancer properties than either of the drug when used alone as evidenced by MTT assay. Besides this, Curcumin and Ellagic acid also restore p53, induce ROS formation and DNA damage. Mechanistic study further indicated that Curcumin and Ellagic acid show anti-HPV activity as evidenced by decrease in the HPV E6 oncoprotein on HeLa cells.

  16. Acid distribution in phosphoric acid fuel cells

    SciTech Connect

    Okae, I.; Seya, A.; Umemoto, M.

    1996-12-31

    Electrolyte acid distribution among each component of a cell is determined by capillary force when the cell is not in operation, but the distribution under the current load conditions had not been clear so far. Since the loss of electrolyte acid during operation is inevitable, it is necessary to store enough amount of acid in every cell. But it must be under the level of which the acid disturbs the diffusion of reactive gases. Accordingly to know the actual acid distribution during operation in a cell is very important. In this report, we carried out experiments to clarify the distribution using small single cells.

  17. Ethacrynic acid oxadiazole analogs induce apoptosis in malignant hematologic cells through downregulation of Mcl-1 and c-FLIP, which was attenuated by GSTP1-1.

    PubMed

    Liu, Guyue; Wang, Rui; Wang, Yuetong; Li, Pengzhan; Zhao, Guisen; Zhao, Linxiang; Jing, Yongkui

    2013-09-01

    Ethacrynic acid, a diuretic, inhibits glutathione S-transferase P1-1 (GSTP1-1) activity and induces cell death in malignant cells at high concentrations. To improve ethacrynic acid activity, ethacrynic acid oxadiazole analogs 6s and 6u were synthesized. Although both compounds have greater antiproliferative effects than ethacrynic acid in human HL-60 cells, 6u has a reduced ability to inhibit GSTP1-1 activity. The mechanisms of both 6s- and 6u-induced cell death as well as the role of GSTP1-1 in their actions were studied. Both 6s and 6u equally induced apoptosis in HL-60 cells due to the activation of caspase-3, -9, and -8, which was correlated with the downregulation of antiapoptotic proteins c-FLIP, Mcl-1, and XIAP. The caspase inhibitor Z-VAD-FMK blocked the reduction of XIAP, but not of c-FLIP and Mcl-1, in 6s-treated cells. The reduction of c-FLIP and Mcl-1 by 6s was not blocked by the proteasomal inhibitor MG132, but was correlated with inhibition of the phosphorylation of extracellular signal-regulated kinase (ERK) and eIF4E. Both 6s and 6u decreased the intracellular glutathione (GSH) levels. N-acetylcysteine blocked reduction in the levels of Mcl-1, c-FLIP, and intracellular GSH as well as apoptosis in HL-60 cells treated by either compound. Silencing of GSTP1-1 in K562 cells sensitized, but overexpression of GSTP1-1 in Raji cells blocked, apoptosis induction by either compound. GSH conjugation at the methylene group abrogated the ability of inducing apoptosis. These data suggest that the methylene group plays an important role in the downregulation of c-FLIP and Mcl-1 proteins and apoptosis induction, which is inactivated by GSTP1-1 by forming GSH conjugates.

  18. Ellagic acid protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

    SciTech Connect

    Ou, Hsiu-Chung; Lee, Wen-Jane; Lee, Shin-Da; Huang, Chih-Yang; Chiu, Tsan-Hung; Tsai, Kun-Ling; Hsu, Wen-Cheng; Sheu, Wayne Huey-Herng

    2010-10-15

    Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized low-density lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. Previous studies have shown that the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase/nitric oxide (PI3K/Akt/eNOS/NO) pathway is involved in oxLDL-induced endothelial apoptosis. Ellagic acid, a natural polyphenol found in berries and nuts, has in recent years been the subject of intense research within the fields of cancer and inflammation. However, its protective effects against oxLDL-induced injury in vascular endothelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. Our results showed that pretreatment with ellagic acid (5-20 {mu}M) significantly attenuated oxLDL-induced cytotoxicity, apoptotic features, and generation of reactive oxygen species (ROS). In addition, the anti-apoptotic effect of ellagic acid was partially inhibited by a PI3K inhibitor (wortmannin) and a specific eNOS inhibitor (cavtratin) but not by an ERK inhibitor (PD98059). In exploring the underlying mechanisms of ellagic acid action, we found that oxLDL decreased Akt and eNOS phosphorylation, which in turn activated NF-{kappa}B and downstream pro-apoptotic signaling events including calcium accumulation, destabilization of mitochondrial permeability, and disruption of the balance between pro- and anti-apoptotic Bcl-2 proteins. Those alterations induced by oxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of oxLDL-induced endothelial apoptosis by ellagic acid is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

  19. EB1 levels are elevated in ascorbic Acid (AA)-stimulated osteoblasts and mediate cell-cell adhesion-induced osteoblast differentiation.

    PubMed

    Pustylnik, Sofia; Fiorino, Cara; Nabavi, Noushin; Zappitelli, Tanya; da Silva, Rosa; Aubin, Jane E; Harrison, Rene E

    2013-07-26

    Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.

  20. LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

    SciTech Connect

    Wang, Huiling; Li, Ridong; Li, Li; Ge, Zemei; Zhou, Rouli; Li, Runtao

    2015-02-27

    In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{sub 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.

  1. Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis.

    PubMed

    Ruggiero, Christine; Elks, Carrie M; Kruger, Claudia; Cleland, Ellen; Addison, Kaity; Noland, Robert C; Stadler, Krisztian

    2014-04-15

    Albuminuria is associated with metabolic syndrome and diabetes. It correlates with the progression of chronic kidney disease, particularly with tubular atrophy. The fatty acid load on albumin significantly increases in obesity, presenting a proinflammatory environment to the proximal tubules. However, little is known about changes in the redox milieu during fatty acid overload and how redox-sensitive mechanisms mediate cell death. Here, we show that albumin with fatty acid impurities or conjugated with palmitate but not albumin itself compromised mitochondrial and cell viability, membrane potential and respiration. Fatty acid overload led to a redox imbalance which deactivated the antioxidant protein peroxiredoxin 2 and caused a peroxide-mediated apoptosis through the redox-sensitive pJNK/caspase-3 pathway. Transfection of tubular cells with peroxiredoxin 2 was protective and mitigated apoptosis. Mitochondrial fatty acid entry and ceramide synthesis modulators suggested that mitochondrial β oxidation but not ceramide synthesis may modulate lipotoxic effects on tubular cell survival. These results suggest that albumin overloaded with fatty acids but not albumin itself changes the redox environment in the tubules, inducing a peroxide-mediated redox-sensitive apoptosis. Thus, mitigating circulating fatty acid levels may be an important factor in both preserving redox balance and preventing tubular cell damage in proteinuric diseases.

  2. PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

    PubMed

    Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2015-10-15

    Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs.

  3. Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells.

    PubMed

    Yeh, Chi-Tai; Yen, Gow-Chin

    2006-05-01

    Phenolic acids have significant biological and pharmacological properties and some have demonstrated remarkable ability to alter sulfate conjugation. However, the modulation mechanisms of phenolic acids on phenol sulfotransferase expression have not been described. In the present study, we investigated the effects of phenolic acids on the expression of the Phase II P-form of phenol sulfotransferase (PST-P) in human hepatoma HepG2 cells. RT-PCR and western blot data revealed that gallic acid induced increase in PST-P expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in PST-P activity. Actinomycin D and cycloheximide inhibited gallic acid-responsive PST-P mRNA expression, indicating that gallic acid is a requirement for transcription and de novo protein synthesis. Transient transfection of HepG2 cells with a reporter plasmid of the upstream region of the human PST gene caused a significant increase in reporter gene activity after gallic acid exposure. Moreover, gallic acid increased the nuclear levels of Nrf2, a transcription factor governing antioxidant response element (ARE). Electrophoretic mobility shift assay showed increased binding of nuclear proteins to ARE consensus sequence after treatment with gallic acid. While investigating the signaling pathways responsible for PST-P induction, we observed that gallic acid activated the p38 mitogen-activated protein kinase (MAPK) pathway. SB203580, a specific inhibitor of p38 MAPK, abolished gallic acid-induced PST-P protein expression. Similarly, gallic acid also caused an accumulation of Nrf2. Moreover, the protective effects of gallic acid on tert-butyl hydroperoxide-induced toxicity was partially blocked by p38 MAPK and PST-P inhibitors, further demonstrating that gallic acid attenuates oxidative stress through a pathway that involves p38 MAPK and PST-P. These results indicate that gallic acid is a potent inducer of PST-P and that PST-P induction is responsible

  4. Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

    SciTech Connect

    Gonzalez-Rubio, Sandra; Linares, Clara I.; Bello, Rosario I.; Gonzalez, Raul; Ferrin, Gustavo; Hidalgo, Ana B.; Munoz-Gomariz, Elisa; Rodriguez, Blanca A.; Barrera, Pilar; Ranchal, Isidora; Duran-Prado, Mario; De la Mata, Manuel; Muntane, Jordi

    2010-01-15

    The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca{sup 2+} on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca{sup 2+} pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca{sup 2+} entrance was induced by A23187 in HepG2. Cell death, Ca{sup 2+} mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca{sup 2+} concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and L-NAME enhanced, GCDCA-induced cell death. The reduction of Ca{sup 2+} entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca{sup 2+} entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca{sup 2+} concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca{sup 2+}-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

  5. Nuclear Raf-1 kinase regulates the CXCR5 promoter by associating with NFATc3 to drive retinoic acid-induced leukemic cell differentiation.

    PubMed

    Geil, Wendy M; Yen, Andrew

    2014-02-01

    Novel functions of signaling molecules have been revealed in studies of cancer stem cells. Retinoic acid (RA) is an embryonic morphogen and stem cell regulator that controls the differentiation of a patient-derived leukemic cell line, HL-60, which is composed of progenitor cells with bipotent myelo-monocytic differentiation capability. RA treatment of HL-60 cells causes unusually long-lasting mitogen-activated protein kinase signaling, with the cells exhibiting the beginning of G0 cell cycle arrest and functional differentiation by 48 h after treatment with RA. This event coincides with the nuclear translocation of Raf-1, phosphorylated at serine 621. The present study shows how the novel localization of Raf-1 to the nucleus results in transcriptional changes that contribute to the differentiation of HL-60 cells induced by RA. We find that nuclear pS621 Raf-1 associates with NFATc3 near its cognate binding site in the promoter of CXCR5, a gene that must be up-regulated to drive RA-induced differentiation. NFATc3 becomes immunoprecipitable with anti-phosphoserine serum, and CXCR5 is transcriptionally up-regulated upon RA-induced differentiation. Inhibiting the pS621 Raf-1/NFATc3 association with PD98059 inhibits these processes and cripples RA-induced differentiation. In this novel paradigm for Raf-1 and RA function, Raf-1 has a role in driving the nuclear signaling of RA-induced differentiation of leukemic progenitor cells.

  6. Perfluorooctanoic acid induces gene promoter hypermethylation of glutathione-S-transferase Pi in human liver L02 cells.

    PubMed

    Tian, Meiping; Peng, Siyuan; Martin, Francis L; Zhang, Jie; Liu, Liangpo; Wang, Zhanlin; Dong, Sijun; Shen, Heqing

    2012-06-14

    Perfluorooctanoic acid (PFOA) is one of the most commonly used perfluorinated compounds. Being a persistent environmental pollutant, it can accumulate in human tissues via various exposure routes. PFOA may interfere in a toxic fashion on the immune system, liver, development, and endocrine systems. In utero human exposure had been associated with cord serum global DNA hypomethylation. In light of this, we investigated possible PFOA-induced DNA methylation alterations in L02 cells in order to shed light into its epigenetic-mediated mechanisms of toxicity in human liver. L02 cells were exposed to 5, 10, 25, 50 or 100 mg/L PFOA for 72h. Global DNA methylation levels were determined by LC/ESI-MS, glutathione-S-transferase Pi (GSTP) gene promoter DNA methylation was investigated by methylation-specific polymerase chain reaction (PCR) with bisulfite sequencing, and consequent mRNA expression levels were measured with quantitative real-time reverse transcriptase PCR. A dose-related increase of GSTP promoter methylation at the transcription factor specificity protein 1 (SP1) binding site was observed. However, PFOA did not significantly influence global DNA methylation; nor did it markedly alter the promoter gene methylation of p16 (cyclin-dependent kinase inhibitor 2A), ERα (estrogen receptor α) or PRB (progesterone receptor B). In addition, PFOA significantly elevated mRNA transcript levels of DNMT3A (which mediates de novo DNA methylation), Acox (lipid metabolism) and p16 (cell apoptosis). Considering the role of GSTP in detoxification, aberrant methylation may be pivotal in PFOA-mediated toxicity response via the inhibition of SP1 binding to GSTP promoter.

  7. Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death.

    PubMed

    Kamijima, Tatsuro; Ohmura, Ayaka; Sato, Toshiya; Akimoto, Kaoru; Itabashi, Miki; Mizuguchi, Mineyuki; Kamiya, Masakatsu; Kikukawa, Takashi; Aizawa, Tomoyasu; Takahashi, Masayuki; Kawano, Keiichi; Demura, Makoto

    2008-11-07

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.

  8. Arachidonic acid release and prostaglandin F(2alpha) formation induced by anandamide and capsaicin in PC12 cells.

    PubMed

    Someya, Akiyoshi; Horie, Syunji; Murayama, Toshihiko

    2002-08-23

    Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.

  9. Effects of Different Simple Triglycerides on Cell Fatty Acid Compositions, Proliferation-Related Protein, and Gene Expressions Induced by Oxidized-LDL in HUVSMCs.

    PubMed

    Li, Hongyan; Pan, Yao; Luo, Kaiyun; Luo, Ting; Fan, Yawei; Deng, Zeyuan

    2017-02-01

    The proliferating effects of 3 different simple triglycerides (tristearin, triolein, and trilinolein) on the human umbilical vein smooth muscle cells (HUVSMCs) induced by oxidized-LDL (ox-LDL) were investigated in this study. The protein and mRNA gene expressions of proliferating cell nuclear antigen (PCNA), smooth muscle-α-actin (SM-α-actin), and monocyte chemoattractant protein-1 (MCP-1) in HUVSMCs were measured by Western blotting and real-time quantitative polymerase chain reaction (PCR). It was shown that in tristearin (SSS) treated HUVSMCs, the saturated fatty acid content was increased, and the compositions of polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid were decreased significantly. On the other hand, triolein (OOO) and trilinolein (LLL) significantly increased the levels of some typical PUFA such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. Moreover, LLL and OOO could upregulate the protein and mRNA expressions of PCNA, MCP-1 as well as downregulate the expression of SM-α-actin. The results also showed that, SSS had significant promotion effects on the proliferation of HUVSMCs induced by ox-LDL. Although both LLL and OOO could inhibit the proliferation of HUVSMCs induced by ox-LDL, and might have certain inhibition of the atherosclerotic process.

  10. MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes.

    PubMed

    Das, Eashita; Bhattacharyya, Nitai Pada

    2014-05-02

    MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0-G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells.

  11. [Effect of APN/CD13 on bestatin enhancing all-trans-retinoic acid-inducing differentiation in NB4 cells].

    PubMed

    Qian, Xi-Jun; Lin, Mao-Fang

    2011-10-01

    This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.

  12. Protective effect of ferulic acid against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative stress in PC12 cells.

    PubMed

    Shen, Y; Zhang, H; Wang, L; Qian, H; Qi, Y; Miao, X; Cheng, L; Qi, X

    2016-01-31

    Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. However, the underlying antioxidant mechanisms of ferulic acid (FA) aganist oxidantive stress are poorly understood. We evaluated the potential protective effects of FA against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced damage in PC12 cells. Our results indicated that pretreatment with FA prior to AAPH exposure significantly increased PC12 cell survival, and also increased catalase and superoxide dismutase activity. Furthermore, FA treatment reduced cellular lactate dehydrogenase release and malondialdehyde levels. It attenuated AAPH-induced apoptosis in PC12 cells, as determined by flow cytometric detection of annexin V. Reductions in mitochondrial membrane potential and accumulation of intracellular Ca2+ were also inhibited by FA treatment. These findings suggested that FA protected PC12 cells against AAPH-induced oxidative stress, and may be a neuroprotective agent.

  13. Protective effects of bupivacaine against kainic acid-induced seizure and neuronal cell death in the rat hippocampus.

    PubMed

    Chiu, Kuan Ming; Wu, Chia Chan; Wang, Ming Jiuh; Lee, Ming Yi; Wang, Su Jane

    2015-01-01

    The excessive release of glutamate is a critical element in the neuropathology of epilepsy, and bupivacaine, a local anesthetic agent, has been shown to inhibit the release of glutamate in rat cerebrocortical nerve terminals. This study investigated whether bupivacaine produces antiseizure and antiexcitotoxic effects using a kainic acid (KA) rat model, an animal model used for temporal lobe epilepsy, and excitotoxic neurodegeneration experiments. The results showed that administering bupivacaine (0.4 mg/kg or 2 mg/kg) intraperitoneally to rats 30 min before intraperitoneal injection of KA (15 mg/kg) increased seizure latency and reduced the seizure score. In addition, bupivacaine attenuated KA-induced hippocampal neuronal cell death, and this protective effect was accompanied by the inhibition of microglial activation and production of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in the hippocampus. Moreover, bupivacaine shortened the latency of escaping onto the platform in the Morris water maze learning performance test. Collectively, these data suggest that bupivacaine has therapeutic potential for treating epilepsy.

  14. Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells.

    PubMed

    Ye, Wenxiu; Hossain, Mohammad Anowar; Munemasa, Shintaro; Nakamura, Yoshimasa; Mori, Izumi C; Murata, Yoshiyuki

    2013-09-01

    We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 μM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.

  15. Elevated systemic glutamic acid level in the non-obese diabetic mouse is Idd linked and induces beta cell apoptosis.

    PubMed

    Banday, Viqar Showkat; Lejon, Kristina

    2017-02-01

    Although type 1 diabetes (T1D) is a T-cell-mediated disease in the effector stage, the mechanism behind the initial beta cell assault is less understood. Metabolomic differences, including elevated levels of glutamic acid, have been observed in patients with T1D before disease onset, as well as in pre-diabetic non-obese diabetic (NOD) mice. Increased levels of glutamic acid damage both neurons and beta cells, implying that this could contribute to the initial events of T1D pathogenesis. We investigated the underlying genetic factors and consequences of the increased levels of glutamic acid in NOD mice. Serum glutamic acid levels from a (NOD×B6)F2 cohort (n = 182) were measured. By genome-wide and Idd region targeted microsatellite mapping, genetic association was detected for six regions including Idd2, Idd4 and Idd22. In silico analysis of potential enzymes and transporters located in and around the mapped regions that are involved in glutamic acid metabolism consisted of alanine aminotransferase, glutamic-oxaloacetic transaminase, aldehyde dehydrogenase 18 family, alutamyl-prolyl-tRNA synthetase, glutamic acid transporters GLAST and EAAC1. Increased EAAC1 protein expression was observed in lysates from livers of NOD mice compared with B6 mice. Functional consequence of the elevated glutamic acid level in NOD mice was tested by culturing NOD. Rag2(-/-) Langerhans' islets with glutamic acid. Induction of apoptosis of the islets was detected upon glutamic acid challenge using TUNEL assay. Our results support the notion that a dysregulated metabolome could contribute to the initiation of T1D. We suggest that targeting of the increased glutamic acid in pre-diabetic patients could be used as a potential therapy.

  16. Interleukin-8 (IL-8) over-production and autocrine cell activation are key factors in monomethylarsonous acid [MMA(III)]-induced malignant transformation of urothelial cells

    SciTech Connect

    Escudero-Lourdes, C.; Wu, T.; Camarillo, J.M.; Gandolfi, A.J.

    2012-01-01

    The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation. -- Highlights: ► IL-8 is over-expressed in human MMA(III)-exposed urothelial

  17. Triglyceride accumulation and fatty acid profile changes in Chlorella (Chlorophyta) during high pH-induced cell cycle inhibition

    SciTech Connect

    Guckert, J.B.; Cooksey, K.E. )

    1990-03-01

    Alkaline pH stress resulted in triglyceride (TG) accumulation in Chlorella CHLOR1 and was independent of medium nitrogen or carbon levels. Based on morphological observations, alkaline pH inhibited autospore release, thus increasing the time for cell cycle completion. Autospore release has been postulated to coincide with TG utilization within the microalgal cell division cycle. The alkaline pH stress affected lipid accumulation by inhibiting the cell division cycle prior to autospore release and, therefore, prior to TG utilization. Cells inhibited in this manner showed an increase in TG accumulation but a decrease in both membrane lipid classes (glycolipid and polar lipid). Unlike TG fatty acid profiles, membrane lipid fatty acid profiles were not stable during TG accumulation. The membrane profiles became similar to the TG, i.e. less unsaturated than in the membrane lipids of unstressed control cells.

  18. Ellagic acid induces cell cycle arrest and apoptosis through TGF-β/Smad3 signaling pathway in human breast cancer MCF-7 cells.

    PubMed

    Chen, Hong-Sheng; Bai, Ming-Han; Zhang, Tao; Li, Guo-Dong; Liu, Ming

    2015-04-01

    Breast cancer represents the second leading cause of cancer-related deaths among women worldwide and preventive therapy could reverse or delay the devastating impact of this disease. Ellagic acid (EA), a dietary flavonoid polyphenol which is present in abundance in pomegranate, muscadine grapes, walnuts and strawberries, has been shown to inhibit cancer cells proliferation and induce apoptosis. Here, we investigated the growth inhibitory effects of EA on MCF-7 breast cancer cells. In the present study, we first found that EA inhibits the proliferation of MCF-7 breast cancer cells mainly mediated by arresting cell cycle in the G0/G1 phase. Moreover, gene expression profiling of MCF-7 breast cancer cell line treated with EA for 6, 12 and 24 h was performed using cDNA microarray. A total of 4,738 genes were found with a >2.0-fold change after 24 h of EA treatment. Among these genes, 2,547 were downregulated and 2,191 were upregulated. Furthermore, the changes of 16 genes, which belong to TGF-β/Smads signaling pathway, were confirmed by real-time RT-PCR and/or western blot analysis. TGF-β/Smads signaling pathway was found as the potential molecular mechanism of EA to regulate breast cancer cell cycle arrest in vitro. Therefore, the regulation of TGF-β/Smads pathway in breast cancer cells could be a novel therapeutic approach for the treatment of patients with breast cancer. Further studies with in vitro models, as well as an analysis of additional human samples, are still needed to confirm the molecular mechanisms of EA in inhibition or prevention of breast cancer growth.

  19. 5,8,11,14-Eicosatetraynoic acid-induced destruction of mitochondria in human prostate cells (PC-3).

    SciTech Connect

    Anderson, K. M.; Seed, T. M.; Wilson, D. E.; Harris, J. E.; Biological and Medical Research; Rush Medical Coll.

    1992-01-01

    Culturing human prostate PC-3 cells for 4, 24, or 72 h in the presence of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of arachidonic acid metabolism and cholesterol biosynthesis, markedly altered the morphology and reduced the number of mitochondria in the treated cells. Using quantitative electron microscopic morphometry, we documented changes in the number, form, area, matrix density, and integrity of the cristae and limiting membranes of mitochondria in cells cultured with ETYA. The inhibition of cholesterol synthesis or the substitution of ETYA for polyunsaturated fatty acids in the inner membrane may participate in the disruption of the mitochondria, which resembles the morphologic sequelae of oxidative stress. If sufficiently extensive, these changes could contribute to the inhibition of cellular proliferation by ETYA.

  20. Pseudolaric acid B-induced autophagy contributes to senescence via enhancement of ROS generation and mitochondrial dysfunction in murine fibrosarcoma L929 cells.

    PubMed

    Qi, Min; Fan, Simiao; Yao, Guodong; Li, Zhao; Zhou, Haiyan; Tashiro, Shin-ichi; Onodera, Satoshi; Xia, Mingyu; Ikejima, Takashi

    2013-01-01

    Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Our previous study demonstrated that PAB induced mitotic catastrophe in L929 cells and indicated that only a small percentage (12%) of the cells undergoing mitotic catastrophe displayed an apoptotic phenotype after PAB treatment for 72 h. In this study, we found that a minority of the cells undergoing mitotic catastrophe ended in apoptosis, and a majority of them entered a period of senescence. Further data confirmed that PAB induced autophagy, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in L929 cells. Subsequently, we found that autophagy inhibitors significantly delayed the senescence process, indicating that autophagy facilitated senescence. Moreover, ROS scavenger significantly decreased the autophagic level and improved mitochondrial function. Additionally, autophagy inhibitors effectively reduced ROS levels and ameliorated mitochondrial function. In conclusion, autophagy promoted senescence via enhancement of ROS generation and mitochondrial dysfunction in PAB-treated L929 cells.

  1. Induction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress.

    PubMed

    Kamalipourazad, Maryam; Sharifi, Mohsen; Maivan, Hassan Zare; Behmanesh, Mehrdad; Chashmi, Najmeh Ahmadian

    2016-10-01

    Manipulation of cell culture media by elicitors is one of most important strategies to inducing secondary metabolism for the production of valuable metabolites. In this investigation, inducing effect of chitosan on physiological, biochemical, and molecular parameters were investigated in cell suspension cultures of Scrophularia striata Boiss. The results showed that chitosan concentration and time of elicitation are determinants of the effectiveness of the elicitor. Accumulation of aromatic amino acids (phenylalanine [Phe] and tyrosine [Tyr]), phenylpropanoid compounds (phenolic acids [PAs] and echinacoside [ECH]), hydrogen peroxide (H2O2) production, phenylalanine ammonia-lyase (PAL) activity and gene expression, and antioxidant enzymes (superoxide dismutase [SOD], peroxidase [POX], catalase [CAT]) activities were altered by changing the exposure time of elicitation. Results showed that, upon elicitation with chitosan, oxidative events were induced, antioxidant responses of S. striata cells were boosted through enhanced activity of an effective series of scavenging enzymes (SOD, CAT, and POX), and biosynthesis of non-enzymatic antioxidants (ECH and PAs [cinnamic, p-coumaric and, caffeic acids]). The increase in amino acid content and PAL activity at early days of exposure to chitosan was related with rises in phenolic compounds. These results provide evidence that chitosan by up-regulation of PAL gene differentially improves the production of phenylpropanoid compounds, which are of medical commercial value with good biotechnological prospects.

  2. Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell

    PubMed Central

    Sohn, Eun Jung; Won, Gunho; Lee, Jihyun; Yoon, Sang Wook; Lee, Ilho; Kim, Hee Jeong; Kim, Sung-Hoon

    2016-01-01

    Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas. PMID:28090191

  3. Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell.

    PubMed

    Sohn, Eun Jung; Won, Gunho; Lee, Jihyun; Yoon, Sang Wook; Lee, Ilho; Kim, Hee Jeong; Kim, Sung-Hoon

    2016-01-01

    Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas.

  4. Protective role of L-ascorbic acid, N-acetylcysteine and apocynin on neomycin-induced hair cell loss in zebrafish.

    PubMed

    Wu, Chia-Yen; Lee, Han-Jung; Liu, Chi-Fang; Korivi, Mallikarjuna; Chen, Hwei-Hsien; Chan, Ming-Huan

    2015-03-01

    Hair cells are highly sensitive to environmental insults and other therapeutic drugs. The adverse effects of drugs such as aminoglycosides can cause hair cell death and lead to hearing loss and imbalance. The objective of the present study was to evaluate the protective activity of L-ascorbic acid, N-acetylcysteine (NAC) and apocynin on neomycin-induced hair cell damage in zebrafish (Danio rerio) larvae at 5 days post fertilization (dpf). Results showed that the loss of hair cells within the neuromasts of the lateral lines after neomycin exposure was evidenced by a significantly lower number of neuromasts labeled with fluorescent dye FM1-43FX observed under a microscope. Co-administration with L-ascorbic acid, NAC and apocynin protected neomycin-induced hair cell loss within the neuromasts. Moreover, these three compounds reduced the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin, indicating that their antioxidant action is involved. In contrast, the neuromasts were labeled with specific fluorescent dye Texas-red conjugated with neomycin to detect neomycin uptake. Interestingly, the uptake of neomycin into hair cells was not influenced by these three antioxidant compounds. These data imply that prevention of hair cell damage against neomycin by L-ascorbic acid, NAC and apocynin might be associated with inhibition of excessive ROS production, but not related to modulating neomycin uptake. Our findings conclude that L-ascorbic acid, NAC and apocynin could be used as therapeutic drugs to protect aminoglycoside-induced listening impairment after further confirmatory studies.

  5. Chlorogenic Acid Combined with Lactobacillus plantarum 2142 Reduced LPS-Induced Intestinal Inflammation and Oxidative Stress in IPEC-J2 Cells

    PubMed Central

    Palócz, Orsolya; Pászti-Gere, Erzsébet; Gálfi, Péter

    2016-01-01

    This study was carried out to investigate protective effect of chlorogenic acid against lipopolysaccharide-induced inflammation and oxidative stress in intestinal epithelial cells. As a marker of inflammatory response, IL-6, IL-8, TNF-α mRNA and protein levels, furthermore, COX-2 mRNA level were followed up. Intracellular redox status and extracellular H2O2 level were also monitored by two fluorescent assays (DCFH-DA, Amplex Red). Moreover, the effect of gut microbiota metabolites in the above mentioned processes was taken into account in our model using Lactobacillus plantarum 2142 bacterial strain. Our data revealed that chlorogenic acid had significant lowering effect on the inflammatory response. Treatment with chlorogenic acid (25–50 μM) significantly decreased gene expression and concentration of proinflammatory cytokines IL-6 and IL-8 compared to LPS-treated cells. COX-2 and TNF-α mRNA levels were also reduced. Furthermore, chlorogenic acid reduced the level of reactive oxygen species in IPEC-J2 cells. Simultaneous application of chlorogenic acid and Lactobacillus plantarum 2142 supernatant resulted protective effect against LPS-induced inflammation and oxidative stress as well. PMID:27861533

  6. Helicteric Acid, Oleanic Acid, and Betulinic Acid, Three Triterpenes from Helicteres angustifolia L., Inhibit Proliferation and Induce Apoptosis in HT-29 Colorectal Cancer Cells via Suppressing NF-κB and STAT3 Signaling

    PubMed Central

    2017-01-01

    Colorectal cancer (CRC) is one of the most common malignancies and most frequent cause of cancer death worldwide. The activation of both NF-κB and STAT3 signaling and the crosstalk between them play an important role in colorectal tumor. Helicteres angustifolia L. is a type of commonly used Chinese medicinal herb and possesses a wide variety of biological activities. In the present study, we investigate the effects of three triterpenes from H. angustifolia (HT) such as helicteric acid (HA), oleanic acid (OA), and betulinic acid (BA), on inhibiting CRC progression. Our results showed that HT extracts could decrease proliferation and induce apoptosis in HT-29 colorectal cancer cells. Moreover, HT extracts could suppress LPS-triggered phosphorylation of IKK, IκB, and NF-κB, attenuate IL-6-induced phosphorylation of JAK2 and STAT3, and suppress the expression of c-Myc, cyclin-D1, and BCL-xL, the downstream gene targets of NF-κB and STAT3. Therefore, HT extracts showed potent therapeutic and antitumor effects on CRC via inhibiting NF-κB and STAT3 signaling. PMID:28331523

  7. Ethacrynic acid butyl-ester induces apoptosis in leukemia cells through a hydrogen peroxide mediated pathway independent of glutathione S-transferase P1-1 inhibition.

    PubMed

    Wang, Rui; Li, Chunmin; Song, Dandan; Zhao, Guisen; Zhao, Linxiang; Jing, Yongkui

    2007-08-15

    Ethacrynic acid (EA), a glutathione S-transferase inhibitor and diuretic agent, inhibits cell growth and induces apoptosis in cancer cells. To improve the activities, the structure of EA has been modified, and it has been shown that EA esters had an increased cell growth inhibitory ability compared with nonesterified analogue. EA butyl-ester (EABE) was synthesized, and its apoptosis induction ability was studied. The efficacy of EABE was compared with that of EA, and the mechanisms of action were studied in HL-60 leukemia cells. EABE exhibited greater cell growth inhibitory and apoptosis induction abilities than did EA. EABE-induced apoptosis in HL-60 cells correlated with increased levels of reactive oxygen species, the death receptor 5 (DR5), and caspase activation and decreased levels of the mitochondrial membrane potential. Pretreatment with antioxidants, either N-acetylcysteine or catalase, completely blocked EABE-induced apoptosis, H2O2 accumulation, and up-regulation of DR5 levels. RG19, a subclone of Raji cells stably transfected with a GSTpi expression vector, and K562 cells with high endogenous GSTP1-1 activity were less sensitive to EABE-induced apoptosis. EABE was more rapidly taken up than EA by HL-60 cells as determined by high-performance liquid chromatography (HPLC) measurements of intracellular concentrations. These results suggest that (a) H2O2 production is a mediator of EABE and EA-induced apoptosis; (b) GSTP1-1 plays a negative role in EABE and EA-induced apoptosis; and (c) the activity of EABE is greater than EA due to its more rapid entry into cells.

  8. The Effects of Topically Applied Glycolic Acid and Salicylic Acid on Ultraviolet Radiation-Induced Erythema, DNA Damage and Sunburn Cell Formation in Human Skin

    PubMed Central

    Kornhauser, Andrija; Wei, Rong-Rong; Yamaguchi, Yuji; Coelho, Sergio G.; Kaidbey, Kays; Barton, Curtis; Takahashi, Kaoruko; Beer, Janusz Z.; Miller, Sharon A.; Hearing, Vincent J.

    2009-01-01

    Background α-Hydroxy acids (αHA) are reported to reduce signs of aging in the skin and are widely used cosmetic ingredients. Several studies suggest that αHA can increase the sensitivity of skin to ultraviolet radiation. More recently, β-hydroxy acids (βHA), or combinations of αHA and βHA have also been incorporated into antiaging skin care products. Concerns have also arisen about increased sensitivity to ultraviolet radiation following use of skin care products containing β-HA. Objective To determine whether topical treatment with glycolic acid, a representative αHA, or with salicylic acid, a βHA, modifies the short-term effects of solar simulated radiation (SSR) in human skin. Methods Fourteen subjects participated in this study. Three of the four test sites on the mid-back of each subject were treated daily Monday - Friday, for a total of 3.5 weeks, with glycolic acid (10%), salicylic acid (2%), or vehicle (control). The fourth site received no treatment. After the last treatment, each site was exposed to SSR, and shave biopsies from all 4 sites were obtained. The endpoints evaluated in this study were erythema (assessed visually and instrumentally), DNA damage and sunburn cell formation. Results Treatment with glycolic acid resulted in increased sensitivity of human skin to SSR, measured as an increase in erythema, DNA damage and sunburn cell formation. Salicylic acid did not produce significant changes in any of these biomarkers. Conclusions Short-term topical application of glycolic acid in a cosmetic formulation increased the sensitivity of human skin to SSR, while a comparable treatment with salicylic acid did not. PMID:19411163

  9. Pinus densiflora Sieb. et Zucc. alleviates lipogenesis and oxidative stress during oleic acid-induced steatosis in HepG2 cells.

    PubMed

    Hwang, Yu-Jin; Wi, Hae-Ri; Kim, Haeng-Ran; Park, Kye Won; Hwang, Kyung-A

    2014-07-23

    Excess accumulation of lipids and oxidative stress in the liver contribute to nonalcoholic fatty liver disease (NAFLD). We hypothesized that Pinus densiflora Sieb. et Zucc. (PSZ) can protect against NAFLD by regulating lipid accumulation and oxidative stress in the liver. To investigate the effect of PSZ upon NAFLD, we used an established cellular model: HepG2 cells treated with oleic acid. Then, the extent of hepatic steatosis and oxidative stress was assessed and levels of inflammatory markers measured. Oleic acid-treated HepG2 cells, compared with controls, had greater lipid accumulation. PSZ decreased lipid accumulation by 63% in oleic acid-treated HepG2 cells. Additionally, PSZ decreased the target gene expression of lipogenesis such as sterol regulatory element binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, diacylglycerol O-acyltransferase-1, and acetyl-CoA carboxylase-1 by 1.75, 6.0, 2.32, 1.93 and 1.81 fold, respectively. In addition, Oleic acid-treated HepG2 cells elicited extensive accumulation of tumor necrosis factor-α (TNFα) by 4.53 fold, whereas PSZ-treated cells decreased the expression of TNFα mRNA by 1.76 fold. PSZ significantly inhibited oxidative stress induced by reactive oxygen species. These results suggest that PSZ has effects on steatosis in vitro and further studies are needed in vivo to verify the current observations.

  10. Pinus densiflora Sieb. et Zucc. Alleviates Lipogenesis and Oxidative Stress during Oleic Acid-Induced Steatosis in HepG2 Cells

    PubMed Central

    Hwang, Yu-Jin; Wi, Hae-Ri; Kim, Haeng-Ran; Park, Kye Won; Hwang, Kyung-A

    2014-01-01

    Excess accumulation of lipids and oxidative stress in the liver contribute to nonalcoholic fatty liver disease (NAFLD). We hypothesized that Pinus densiflora Sieb. et Zucc. (PSZ) can protect against NAFLD by regulating lipid accumulation and oxidative stress in the liver. To investigate the effect of PSZ upon NAFLD, we used an established cellular model: HepG2 cells treated with oleic acid. Then, the extent of hepatic steatosis and oxidative stress was assessed and levels of inflammatory markers measured. Oleic acid-treated HepG2 cells, compared with controls, had greater lipid accumulation. PSZ decreased lipid accumulation by 63% in oleic acid-treated HepG2 cells. Additionally, PSZ decreased the target gene expression of lipogenesis such as sterol regulatory element binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, diacylglycerol O-acyltransferase-1, and acetyl-CoA carboxylase-1 by 1.75, 6.0, 2.32, 1.93 and 1.81 fold, respectively. In addition, Oleic acid-treated HepG2 cells elicited extensive accumulation of tumor necrosis factor-α (TNFα) by 4.53 fold, whereas PSZ-treated cells decreased the expression of TNFα mRNA by 1.76 fold. PSZ significantly inhibited oxidative stress induced by reactive oxygen species. These results suggest that PSZ has effects on steatosis in vitro and further studies are needed in vivo to verify the current observations. PMID:25057104

  11. Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

    SciTech Connect

    Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

    2013-08-02

    Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis.

  12. Heat shock protein 27 protects against aminolevulinic acid-mediated photodynamic therapy-induced apoptosis and necrosis in human breast cancer cells.

    PubMed

    Ziegler, Sarah A; Loucks, Cherisse; Madsen, Steen J; Carper, Stephen W

    2007-01-01

    This study utilized two breast cancer cell lines differing only in their expression of heat shock protein 27 (hsp27). The DB46 cell line was engineered to express high constitutive levels of hsp27, while the DC4 cell line expresses normal low levels of hsp27. The cells were incubated in 1 mM aminolevlinic acid (ALA) 4 hr prior to light exposures (635 nm) ranging from 1 to 20 J/cm2. Both cell lines displayed a dose response to photodynamic therapy (PDT) as assayed by clonogenic survival. LD50s of 2.68 and 1.27 J/cm2 were observed for DB46 and DC4 cells respectively. ALA-PDT-induced resistance to both apoptosis and necrosis in the DB46 cell line was found from TUNEL assays and fluorescence microscopy studies using propidium iodide and Hoechst staining.

  13. Zoledronic acid induces dose-dependent increase of antigen-specific CD8 T-cell responses in combination with peptide/poly-IC vaccine.

    PubMed

    Park, Hye-Mi; Cho, Hyun-Il; Shin, Chang-Ae; Shon, Hyun-Jung; Kim, Tai-Gyu

    2016-03-04

    Zoledronic acid (ZA) is used for treating osteoporosis and for preventing skeletal fractures in cancer patients suffering from myeloma and prostate cancer. It is also reported to directly induce cancer cell apoptosis and indirectly modulate T-cell immune response as an antitumor agent. In this study, the effect of ZA following peptide/polyinosinic-polycytidylic acid (poly-IC) vaccination was investigated in a murine tumor model. The combination of ZA with peptide/poly-IC vaccine showed a synergistic effect on the induction of antigen-specific CD8 T-cell response. Three consecutive intravenous administrations of ZA was defined to induce the highest CD8 T-cell response. Further, total splenocyte counts and antigen-specific CD8 T-cell response gradually increased depending on the dose of ZA. In tumor-bearing mice, ZA showed a dose-dependent decrease of growth and prolonged survival. Treatment with ZA only decreased the number of CD11b(+)Gr1(+) myeloid cells in blood. Our results demonstrate that the use of ZA could improve antitumor immune responses induced by the peptide/poly-IC vaccine.

  14. Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy.

    PubMed

    Eriksen, Agnete Bratsberg; Torgersen, Maria Lyngaas; Holm, Kristine Lillebø; Abrahamsen, Greger; Spurkland, Anne; Moskaug, Jan Øivind; Simonsen, Anne; Blomhoff, Heidi Kiil

    2015-01-01

    In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9- and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RA-stimulated IgG production in TLR9- and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system.

  15. Metformin-induced killing of triple-negative breast cancer cells is mediated by reduction in fatty acid synthase via miRNA-193b.

    PubMed

    Wahdan-Alaswad, Reema S; Cochrane, Dawn R; Spoelstra, Nicole S; Howe, Erin N; Edgerton, Susan M; Anderson, Steven M; Thor, Ann D; Richer, Jennifer K

    2014-12-01

    The anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) reduces both the incidence and mortality of several types of cancer. Metformin has been shown to selectively kill cancer stem cells, and triple-negative breast cancer (TNBC) cell lines are more sensitive to the effects of metformin as compared to luminal breast cancer. However, the mechanism underlying the enhanced susceptibility of TNBC to metformin has not been elucidated. Expression profiling of metformin-treated TNBC lines revealed fatty acid synthase (FASN) as one of the genes most significantly downregulated following 24 h of treatment, and a decrease in FASN protein was also observed. Since FASN is critical for de novo fatty acid synthesis and is important for the survival of TNBC, we hypothesized that FASN downregulation facilitates metformin-induced apoptosis. Profiling studies also exposed a rapid metformin-induced increase in miR-193 family members, and miR-193b directly targets the FASN 3'UTR. Addition of exogenous miR-193b mimic to untreated TNBC cells decreased FASN protein expression and increased apoptosis of TNBC cells, while spontaneously immortalized, non-transformed breast epithelial cells remained unaffected. Conversely, antagonizing miR-193 activity impaired the ability of metformin to decrease FASN and cause cell death. Further, the metformin-stimulated increase in miR-193 resulted in reduced mammosphere formation by TNBC lines. These studies provide mechanistic insight into metformin-induced killing of TNBC.

  16. Lysophosphatidic acid induces both EGFR-dependent and EGFR-independent effects on DNA synthesis and migration in pancreatic and colorectal carcinoma cells.

    PubMed

    Tveteraas, Ingun Heiene; Aasrum, Monica; Brusevold, Ingvild Johnsen; Ødegård, John; Christoffersen, Thoralf; Sandnes, Dagny

    2016-02-01

    Lysophosphatidic acid (LPA) is a small glycerophospholipid ubiquitously present in tissues and plasma. It acts through receptors belonging to the G-protein-coupled receptor (GPCR) family, is involved in several biological processes, and is strongly implicated in different cancers. In this paper, we have investigated the effects of LPA on DNA synthesis and migration in a panel of pancreatic and colon cancer cells, with particular focus on the involvement of the epidermal growth factor (EGF) receptor (EGFR) in LPA-induced signaling. LPA stimulated DNA synthesis and/or migration in all the cell lines included in this study. In five of the six cell lines, LPA induced phosphorylation of the EGFR, and the effects on EGFR and Akt, and in some of the cells also ERK, were sensitive to the EGFR tyrosine kinase inhibitor gefitinib, strongly suggesting LPA-induced EGFR transactivation in these cells. In contrast, in one of the pancreatic carcinoma cell lines (Panc-1), we found no evidence of transactivation of the EGFR. In the pancreatic carcinoma cell lines where transactivation took place (BxPC3, AsPC1, HPAFII), gefitinib reduced LPA-induced DNA synthesis and/or migration. However, we also found evidence of transactivation in the two colon carcinoma cell lines (HT29, HCT116) although gefitinib did not inhibit LPA-induced DNA synthesis or migration in these cells. Taken together, the data indicate that in many gastrointestinal carcinoma cells, LPA uses EGFR transactivation as a mechanism when exerting such effects as stimulation of cell proliferation and migration, but EGFR-independent pathways may be involved instead of, or in concerted action with, the EGFR transactivation.

  17. The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR

    PubMed Central

    Zhang, Xu Hannah; Zhao, Chunying; Ma, Zhongmin Alex

    2010-01-01

    Summary The G1 phase of the cell cycle is marked by the rapid turnover of phospholipids. This turnover is regulated by CTP:phosphocholine-cytidylyltransferase (CCT) and group VIA Ca2+-independent-phospholipase A2 (iPLA2). We previously reported that inhibition of iPLA2 arrests cells in G1 phase of the cell cycle by activating the p53-p21 checkpoint. Here we further characterize the mechanism of p53 activation. We show that specific inhibition of iPLA2 induces a time dependent phosphorylation of Ser15 in p53 in the absence of DNA damage. This phosphorylation requires the kinase ataxia-telangiectasia and Rad-3-related (ATR) but not the ataxia-telangiectasia-mutated (ATM) kinase. Moreover, we identify in cell membranes a significant increase of phosphatidylcholines (PCs) containing chains of polyunsaturated fatty acids and a decrease of PCs containing saturated fatty acids in response to inhibition of iPLA2. The time course of phosphorylation of Ser15 in p53 correlates with increasing levels of PCs containing polyunsaturated fatty acids. We further demonstrate that the PCs with linoleic acid in their sn-2 position (18:2n6) induce phosphorylation of Ser15 in p53 in an ATR-dependent manner. Our findings establish that cells can regulate the levels of polyunsaturated fatty acids in phospholipids through iPLA2-mediated deacylation of PCs. Disruption of this regulation increases the proportions of PCs containing polyunsaturated fatty acids and activates the ATR-p53 signalling pathway. PMID:18032786

  18. Assessment of the chemical changes induced in human melanoma cells by boric acid treatment using infrared imaging

    SciTech Connect

    Acerbo, A.; Miller, L.

    2009-07-01

    Boron is found in everyday foods and drinking water in trace quantities. Boron exists as boric acid (BA) within plants and animals, where low levels have been linked to cancer incidence. However, this correlation is not well characterized. In this study, we examined the chemical and morphological effects of BA on human skin melanoma cells (SK-MEL28) using Fourier Transform InfraRed Imaging (FTIRI) with a Focal Plane Array (FPA) detector. Cells were grown under concentrations of BA ranging from 0 to 50 mM. Cell viability was determined after 1, 2, 3, 5, 7 and 10 days using trypan blue staining. With FTIRI, images of approximately twenty cells per time point per condition were collected. Principal components analysis (PCA) was used to evaluate changes in cell composition, with particular focus on the lipid, protein, and nucleic acid spectral components. Results from trypan blue staining revealed decreased cell viability as BA concentration increased. FTIRI data indicated that the protein and lipid contents (as indicated by the lipid/protein ratio) did not undergo substantial changes due to BA treatment. In contrast, the nucleic acid/protein ratio significantly decreased with BA treatment. PCA results showed an increase in {beta}-sheet protein at higher concentrations of BA (12.5, 25, and 50 mM). Together, these results suggest that high concentrations of BA have an anti-proliferative effect and show signs consistent with apoptosis.

  19. Assessment of the chemical changes induced in human melanoma cells by boric acid treatment using infrared imaging.

    PubMed

    Acerbo, Alvin S; Miller, Lisa M

    2009-08-01

    Boron is found in everyday foods and drinking water in trace quantities. Boron exists as boric acid (BA) within plants and animals, where low levels have been linked to cancer incidence. However, this correlation is not well characterized. In this study, we examined the chemical and morphological effects of BA on human skin melanoma cells (SK-MEL28) using Fourier Transform InfraRed Imaging (FTIRI) with a Focal Plane Array (FPA) detector. Cells were grown under concentrations of BA ranging from 0 to 50 mM. Cell viability was determined after 1, 2, 3, 5, 7 and 10 days using trypan blue staining. With FTIRI, images of approximately twenty cells per time point per condition were collected. Principal components analysis (PCA) was used to evaluate changes in cell composition, with particular focus on the lipid, protein, and nucleic acid spectral components. Results from trypan blue staining revealed decreased cell viability as BA concentration increased. FTIRI data indicated that the protein and lipid contents (as indicated by the lipid/protein ratio) did not undergo substantial changes due to BA treatment. In contrast, the nucleic acid/protein ratio significantly decreased with BA treatment. PCA results showed an increase in beta-sheet protein at higher concentrations of BA (12.5, 25, and 50 mM). Together, these results suggest that high concentrations of BA have an anti-proliferative effect and show signs consistent with apoptosis.

  20. Ligustrazine-Oleanolic Acid Glycine Derivative, G-TOA, Selectively Inhibited the Proliferation and Induced Apoptosis of Activated HSC-T6 Cells.

    PubMed

    Bi, Siling; Chu, Fuhao; Wang, Mina; Li, Bi; Mao, Pei; Zhang, Huazheng; Wang, Penglong; Guo, Wenbo; Xu, Liang; Ren, Liwei; Lei, Haimin; Zhang, Yuzhong

    2016-11-23

    Hepatic fibrosis is a naturally occurring wound-healing reaction, with an imbalance of extracellular matrix (ECM) during tissue repair response, which can further deteriorate to hepatocellular carcinoma without timely treatment. Inhibiting activated hepatic stellate cell (HSC) proliferation and inducing apoptosis are the main methods for the treatment of liver fibrosis. In our previous study, we found that the TOA-glycine derivative (G-TOA) had exhibited more significant inhibitory activity against HepG2 cells and better hydrophilicity than TOA, ligustrazine (TMP), and oleanolic acid (OA). However, inhibiting activated HSC proliferation and inducing apoptosis by G-TOA had not been reported. In this paper, the selective cytotoxicity of G-TOA was evaluated on HSC-T6 cells and L02 cells, and apoptosis mechanisms were explored. It was found that G-TOA could selectively inhibit the proliferation of activated HSC-T6 cells, induce morphological changes, early apoptosis, and mitochondrial membrane potential depolarization, increase intracellular free calcium levels, downregulate the expression of NF-κB/p65 and COX-2 protein, and decrease the ratio of Bcl-2/Bax, thereby inducing HSC-T6 cell apoptosis. Thence, G-TOA might be a potential antifibrosis agent for the therapy of hepatic fibrosis, provided that it exerts anti-fibrosis effects on activated HSC-T6 cells.

  1. Docosapentaenoic acid (22:5, n-3) suppressed tube-forming activity in endothelial cells induced by vascular endothelial growth factor.

    PubMed

    Tsuji, Masako; Murota, Se-itsu; Morita, Ikuo

    2003-05-01

    It is generally accepted that n-3 polyunsaturated fatty acids have beneficial effects on vascular homeostasis. Among the several functions of endothelial cells, angiogenesis contributes to tumor growth, inflammation, and microangiopathy. We have already demonstrated that eicosapentaenoic acid (EPA, 20:5, n-3) suppressed angiogenesis. In this paper, we examined the effect of docosapentaenoic acid (DPA, 22:5, n-3), an elongated metabolite of EPA, on tube-forming activity in bovine aortic endothelial cells (BAE cells) incubated between type I collagen gels. The pretreatment of BAE cells with DPA suppressed tube-forming activity induced by vascular endothelial growth factor (VEGF). The effect of DPA was stronger than those of EPA and docosahexaenoic acid (22:6, n-3). The migrating activity of endothelial cells stimulated with VEGF was also suppressed by DPA pretreatment. The treatment of BAE cells with DPA caused the suppression of VEGF receptor-2 (VEGFR-2, the kinase insert domain-containing receptor, KDR) expression in both plastic dish and collagen gel cultures. These data indicate that DPA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 expression.

  2. Docosahexaenoic acid inhibits vascular endothelial growth factor (VEGF)-induced cell migration via the GPR120/PP2A/ERK1/2/eNOS signaling pathway in human umbilical vein endothelial cells.

    PubMed

    Chao, Che-Yi; Lii, Chong-Kuei; Ye, Siou-Yu; Li, Chien-Chun; Lu, Chia-Yang; Lin, Ai-Hsuan; Liu, Kai-Li; Chen, Haw-Wen

    2014-05-07

    Cell migration plays an important role in angiogenesis and wound repair. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is essential for endothelial cell survival, proliferation, and migration. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, shows both anti-inflammatory and antioxidant activities in vitro and in vivo. This study investigated the molecular mechanism by which DHA down-regulates VEGF-induced cell migration. HUVECs were used as the study model, and the MTT assay, Western blot, wound-healing assay, and phosphatase activity assay were used to explore the effects of DHA on cell migration. GPR120 is the putative receptor for DHA action. The results showed that DHA, PD98059 (an ERK1/2 inhibitor), and GW9508 (a GPR120 agonist) inhibited VEGF-induced cell migration. In contrast, pretreatment with okadaic acid (OA, a PP2A inhibitor) and S-nitroso-N-acetyl-DL-penicillamine (an NO donor) reversed the inhibition of cell migration by DHA. VEGF-induced cell migration was accompanied by phosphorylation of ERK1/2 and eNOS. Treatment of HUVECs with DHA increased PP2A enzyme activity and decreased VEGF-induced phosphorylation of ERK1/2 and eNOS. However, pretreatment with OA significantly decreased DHA-induced PP2A enzyme activity and reversed the DHA inhibition of VEGF-induced ERK1/2 and eNOS phosphorylation. These results suggest that stimulation of PP2A activity and inhibition of the VEGF-induced ERK1/2/eNOS signaling pathway may be involved in the DHA suppression of VEGF-induced cell migration. Thus, the effect of DHA on angiogenesis and wound repair is at least partly by virtue of its attenuation of cell migration.

  3. Morphologic Damage of Rat Alveolar Epithelial Type II Cells Induced by Bile Acids Could Be Ameliorated by Farnesoid X Receptor Inhibitor Z-Guggulsterone In Vitro

    PubMed Central

    Huang, Yaowei; Hou, Xusheng; Wu, Wenyu; Nie, Lei; Tian, Yinghong; Lu, Yanmeng

    2016-01-01

    Objective. To determine whether bile acids (BAs) affect respiratory functions through the farnesoid X receptor (FXR) expressed in the lungs and to explore the possible mechanisms of BAs-induced respiratory disorder. Methods. Primary cultured alveolar epithelial type II cells (AECIIs) of rat were treated with different concentrations of chenodeoxycholic acid (CDCA) in the presence or absence of FXR inhibitor Z-guggulsterone (GS). Then, expression of FXR in nuclei of AECIIs was assessed by immunofluorescence microscopy. And ultrastructural changes of the cells were observed under transmission electron microscope and analyzed by Image-Pro Plus software. Results. Morphologic damage of AECIIs was exhibited in high BAs group in vitro, with high-level expression of FXR, while FXR inhibitor GS could attenuate the cytotoxicity of BAs to AECIIs. Conclusions. FXR expression was related to the morphologic damage of AECIIs induced by BAs, thus influencing respiratory functions. PMID:27340672

  4. AKT/mTOR signaling pathway is involved in salvianolic acid B-induced autophagy and apoptosis in hepatocellular carcinoma cells.

    PubMed

    Gong, Ling; Di, Chunhong; Xia, Xiaofang; Wang, Jie; Chen, Gongying; Shi, Junping; Chen, Pengshuai; Xu, Hui; Zhang, Weibing

    2016-12-01

    Chinese medicines are emerging as an attractive new generation of anticancer drugs. Here, we explored the impact of salvianolic acid B (Sal B), the major water-soluble compounds of Danshen, on apoptosis and autophagy of human hepatocellular carcinoma cells (HCC). We also investigated the related molecular mechanisms. We found that Sal B exhibits potent ability to inhibit HCC cells viability in a concentration-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, Sal B could also induce autophagy. Furthermore, pretreatment with the autophagy inhibitor chloroquine or 3-methyladenine showed the potential in attenuating the apoptosis rate induced by Sal B. Mechanistically, Sal B treatment inhibited the AKT/mTOR signaling cascade in vitro. Overexpression of AKT abolished the effects of Sal B on HCC cells, suggesting a critical role of the AKT/mTOR signaling pathway in Sal B-induced biological effects. Our results indicated that the mitochondrial pathway was involved in Sal B-induced apoptosis of HCC cells. Moreover, the AKT/mTOR signaling pathway was involved in Sal B-induced autophagy, which promoted apoptosis. This study may provide a promising strategy for using Sal B as a chemotherapeutic agent for patients with HCC.

  5. The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species

    PubMed Central

    Ruefli, Astrid A.; Ausserlechner, Michael J.; Bernhard, David; Sutton, Vivien R.; Tainton, Kellie M.; Kofler, Reinhard; Smyth, Mark J.; Johnstone, Ricky W.

    2001-01-01

    Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death. PMID:11535817

  6. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

    SciTech Connect

    Zhang, Yuanyuan; Han, Lirong; Qi, Wentao; Cheng, Dai; Ma, Xiaolei; Hou, Lihua; Cao, Xiaohong; Wang, Chunling

    2015-01-24

    Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP

  7. Flow cytometric assessment of cell structural and functional changes induced by acetic acid in the yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae.

    PubMed

    Prudêncio, C; Sansonetty, F; Côrte-Real, M

    1998-04-01

    Flow cytometry (FCM) was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii (AA resistant) and Saccharomyces cerevisiae (AA sensitive). Kinetic changes in esterase activity, intracellular dye processing, and membrane integrity were monitored, and to detect those changes we used three assays involving fluorescein diacetate hydrolysis, FUN-1 processing, and propidium iodide exclusion, respectively. In S. cerevisiae, the decrease in the ability to process FUN-1 preceded the decrease in esterase activity, and there was loss of cell membrane integrity after incubation with AA. In Z. bailii, with higher AA concentrations, there was a similar decrease in the ability to process FUN-1, which also preceded the loss of cell membrane integrity. Changes in esterase activity in this yeast induced by AA treatment could not be monitored because the changes occurred independently of the presence of the acid. For control samples (untreated cells killed with 10% v/v of AA), the percentages of nonaltered cells as estimated by FCM and percentages of viable cells as estimated by colony forming unit (CFU) counts were identical. However, for cell samples treated for short periods with 3% (v/v) or less of AA, none of the dyes produced FCM results comparable to those produced by CFU counts.

  8. Superoxide anions produced by inflammatory cells play an important part in the pathogenesis of acid and pepsin induced oesophagitis in rabbits.

    PubMed Central

    Naya, M J; Pereboom, D; Ortego, J; Alda, J O; Lanas, A

    1997-01-01

    BACKGROUND: Reactive oxygen metabolites have been associated with gastrointestinal injury. OBJECTIVE: To investigate whether mucosal reactive oxygen metabolites are involved in acid and pepsin induced oesophagitis, and if so, which specific metabolites. METHODS: The effects of free radical scavengers and the anti-inflammatory drug ketotifen on rabbit oesophagitis induced by acidified pepsin were studied. Isolated oesophageal cells were obtained before and after oesophageal injury and the generation of superoxide anion and hydrogen peroxide was analysed by flow cytometry. The presence of inflammatory cells was determined by indirect immunofluorescence with a mouse antirabbit CD11b antibody. RESULTS: Of the free radical scavengers tested, superoxide dismutase, which reacts with the superoxide anion, significantly reduced oesophagitis, whereas catalase, which reacts with hydrogen peroxide, had only a mild effect and dimethylsulphoxide had no effect. Ketotifen significantly reduced the inflammation and also prevented the induction of oesophagitis. Isolated cells obtained from the oesophageal mucosa after acidified pepsin exposure generated increased amounts of superoxide anions, which were mainly produced by CD11b positive cells. CONCLUSIONS: Reactive oxygen metabolites, especially superoxide anion, produced by inflammatory cells play a significant part in the genesis of oesophagitis induced by acid and pepsin in rabbits and might be a target for future medical therapy. Images PMID:9071927

  9. Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

    PubMed

    Kucherenko, Yuliya V; Lang, Florian

    2014-07-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

  10. Different effects of bile acids, ursodeoxycholic acid and deoxycholic acid, on cell growth and cell death in human colonic adenocarcinoma cells.

    PubMed

    Shiraki, Katsuya; Ito, Takeshi; Sugimoto, Kazushi; Fuke, Hiroyuki; Inoue, Tomoko; Miyashita, Kazumi; Yamanaka, Takenari; Suzuki, Masahiro; Nabeshima, Kazuo; Nakano, Takeshi; Takase, Koujiro

    2005-10-01

    Secondary bile acids have been implicated as an important etiological factor in colorectal cancer. We investigated the effects of ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) on the growth and cytotoxicity in HT29 human colonic adenocarcinoma cells. Proliferation assay, cell cycle analysis and cell death characterization by bile acids were performed. Both UDCA and DCA reduced their proliferation rate of HT29 over 48 h in a concentration- and time-dependent manner compared with control cultures. In terms of cell cycle effects, however, UDCA induced G2/M arrest, while DCA induced G1 arrest in a concentration- and time-dependent manner. As for the effects of each bile acid on cell toxicity, UDCA induced early apoptosis and DCA induced both early apoptosis and necrosis. Bile acids play an important role in regulating cell survival and cell death in colon adenocarcinoma cells.

  11. Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes

    SciTech Connect

    Chang, L.W.; Daniel, F.B. ); DeAngelo, A.B. )

    1992-01-01

    An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.

  12. All-Trans-Retinoid Acid Induces the Differentiation of Encapsulated Mouse Embryonic Stem Cells into GABAergic Neurons

    PubMed Central

    Addae, Cynthia; Yi, Xiaoping; Gernapudi, Ramkishore; Cheng, Henrique; Musto, Alberto; Martinez-Ceballos, Eduardo

    2012-01-01

    Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that 1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and 2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (~87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders. PMID:22466603

  13. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  14. A minority of carcinoma cells producing acidic fibroblast growth factor induces a community effect for tumor progression.

    PubMed Central

    Jouanneau, J; Moens, G; Bourgeois, Y; Poupon, M F; Thiery, J P

    1994-01-01

    It is generally accepted that primary tumors become heterogeneous as a consequence of tumor-cell genetic instability. Clonal dominance has been shown to occur in some experimental models allowing a subpopulation of cells to overgrow the primary heterogeneous tumor and to metastasize. Alternatively, interactions among coexisting tumor subpopulations may contribute to the emergence of a malignant invasive primary solid tumor. We asked the question whether emergence of carcinoma cells producing a growth/dissociating factor within a tumor cell population may be a determinant for tumor progression and for clonal dominance. To mimic such a situation, we have investigated the impact of tumor subpopulation heterogeneity in an in vivo model in which mixtures of carcinoma cells that differ in their ability to produce acidic fibroblast growth factor are injected into nude mice. Our data indicate that a growth-factor-producing cell subpopulation can confer increased tumorigenicity to an entire cell population and subsequently elicit a shorter delay for appearance of metastasis. A community effect via cell interactions may account for a heterogeneous tumor cell population rather than clonal dominance during progression of certain tumor types. Images Fig. 3 PMID:7506417

  15. Tamoxifen enhances the differentiation-inducing and growth-inhibitory effects of all-trans retinoic acid in acute promyelocytic leukemia cells.

    PubMed

    Adachi, Koji; Honma, Yoshio; Miyake, Takaaki; Kawakami, Koshi; Takahashi, Tsutomu; Suzumiya, Junji

    2016-03-01

    All-trans retinoic acid (ATRA) is valuable in differentiation therapy for acute promyelocytic leukemia (APL). However, ATRA has had limited success as a single agent, due to the development of resistance. We found that tamoxifen effectively enhanced the differentiation-inducing effect of ATRA. Tamoxifen alone inhibited the proliferation of myeloid leukemia cell lines while only slightly increasing morphologic differentiation. Tamoxifen effectively enhanced the growth-inhibiting actions of various differentiation-inducing agents. ATRA in the presence of tamoxifen increased NBT reduction and the expression of CD11b in HL-60 cells more effectively than ATRA alone. Tamoxifen also enhanced the differentiation induced by the other inducers tested. ATRA induced the differentiation of APL cell lines NB4 and HT93 and APL cells in primary culture, and this differentiation was also enhanced by tamoxifen. Tamoxifen is one of the most widely used drugs for the treatment of cancer and has few side effects. The combination of ATRA and tamoxifen might be considered for the treatment of APL patients in whom it can be difficult to apply arsenic trioxide or anthracyclines.

  16. Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line

    PubMed Central

    Kogai, Takahiko; Schultz, James J.; Johnson, Laura S.; Huang, Min; Brent, Gregory A.

    2000-01-01

    The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to ≈9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein (≈68 kDa). tRA stimulated NIS gene transcription ≈4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t1/2 = 24 min), compared with that in FRTL-5 thyroid cells (t1/2 = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with 131I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers. PMID:10890895

  17. Cytotoxic effect of p-Coumaric acid on neuroblastoma, N2a cell via generation of reactive oxygen species leading to dysfunction of mitochondria inducing apoptosis and autophagy.

    PubMed

    Shailasree, S; Venkataramana, M; Niranjana, S R; Prakash, H S

    2015-02-01

    p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. In the present study, p-CA was evaluated for its capacity to induce cytotoxic effect to neuroblastoma N2a cells and we report here the possible mechanism of its action. p-CA at a concentration of 150 μmol/L, upon exposure for 72 h, stimulated 81.23 % of cells to apoptosis, as evidenced by flow cytometer studies mediated through elevated levels of ROS (7.5-fold over control). Excess ROS production activated structural injury to mitochondrial membrane, observed as dissipation of its membrane potential and followed by the release of cytochrome c (8.73-fold). Enhanced generation of intracellular ROS correlated well with the decreased levels (~60 %) of intracellular GSH. Sensitizing neuroblastoma cells for induction of apoptosis by p-CA identified p53-mediated upregulated accumulation of caspase-8 messenger RNA (2.8-fold). Our data report on autophagy, representing an additional mechanism of p-CA to induce growth arrest, detected by immunoblotting and fluorescence, correlated with accumulation of elevated levels (1.2-fold) of the LC3-II protein and acridine orange-stained autophagosomes, both autophagy markers. The present study indicates p-CA was effective in production of ROS-dependent mitochondrial damage-induced cytotoxicity in N2a cells.

  18. Bitter gourd seed fatty acid rich in 9c,11t,13t-conjugated linolenic acid induces apoptosis and up-regulates the GADD45, p53 and PPARgamma in human colon cancer Caco-2 cells.

    PubMed

    Yasui, Yumiko; Hosokawa, Masashi; Sahara, Takehiko; Suzuki, Rikako; Ohgiya, Satoru; Kohno, Hiroyuki; Tanaka, Takuji; Miyashita, Kazuo

    2005-08-01

    Bitter gourd (Momordica charantia) seed oil (BGO) is a unique oil which contains 9cis, 11trans, 13trans-conjugated linolenic acid (9c,11t,13t-CLN) at a high level of more than 60%. In this study, we investigated the anti-proliferative and apoptosis-inducing effects of free fatty acids prepared from BGO (BGO-FFA) using colon cancer Caco-2 cells. BGO-FFA and purified 9c,11t,13t-CLN remarkably reduced the cell viability of Caco-2. In Caco-2 cells treated with BGO-FFA, DNA fragmentation of apoptosis indicators was observed in a dose-dependent manner. The expression level of apoptosis suppressor Bcl-2 protein was also decreased by BGO-FFA treatment. The GADD45 and p53, which play an important role in apoptosis-inducing pathways, were remarkably up-regulated by BGO-FFA treatment in Caco-2 cells. Up-regulation of PPARgamma mRNA and protein were also observed during apoptosis induced by BGO-FFA. These results suggest that BGO-FFA rich in 9c,11t,13t-CLN may induce apoptosis in Caco-2 cells through up-regulation of GADD45, p53 and PPARgamma.

  19. Atomic evidence that modification of H-bonds established with amino acids critical for host-cell binding induces sterile immunity against malaria

    SciTech Connect

    Patarroyo, Manuel E.; Cifuentes, Gladys; Pirajan, Camilo; Moreno-Vranich, Armando; Vanegas, Magnolia

    2010-04-09

    Based on the 3D X-ray crystallographic structures of relevant proteins of the malaria parasite involved in invasion to host cells and 3D NMR structures of High Activity Binding Peptides (HABPs) and their respective analogues, it was found that HABPs are rendered into highly immunogenic and sterile immunity inducers in the Aotus experimental model by modifying those amino acids that establish H-bonds with other HABPs or binding to host's cells. This finding adds striking and novel physicochemical principles, at the atomic level, for a logical and rational vaccine development methodology against infectious disease, among them malaria.

  20. MEMANTINE ATTENUATES THE OKADAIC ACID INDUCED SHORT-TERM SPATIAL MEMORY IMPAIRMENT AND HIPPOCAMPAL CELL LOSS IN RATS.

    PubMed

    Dashniani, M; Chighladze, M; Burjanadze, M; Beselia, G; Kruashvili, L

    2016-03-01

    In the present study, the possible beneficial effect of memantine on the Okadaic Acid (OA) induced spatial short-term memory impairment was examined in spatial alternation task, and the neuroprotective potential of memantine on OA-induced structural changes in the hippocampus was evaluated by Nissl staining. OA was dissolved in artificial cerebrospinal fluid (aCSF) and injected intracerebroventriculary (ICV) 200 ng in a volume of 10 μl bilaterally. Vehicle control received aCSF ICV bilaterally. Control and OA injected rats were divided into 2 subgroups injected i.p. with saline or memantine (5 mg/kg). Memantine or saline were given daily for 13 days starting from the day of OA injection. Behavioral study showed that bilateral ICV microinjection of OA induced impairment in spatial short-term memory. Nissl staining in the present study showed that the ICV microinjection of OA significantly decreased the number of surviving pyramidal neurons in the CA1 region of the hippocampus. Chronic administration of memantine effectively attenuated OA induced spatial short-term memory impairment and the OA-induced neuropathological changes in the hippocampus. Therefore, ICV injection of OA can be used as an experimental model to study mechanisms of neurodegeneration and define novel therapeutics targets for AD pathology.

  1. Folic acid protects against lead acetate-induced hepatotoxicity by decreasing NF-κB, IL-1β production and lipid peroxidation mediataed cell injury.

    PubMed

    Abd Allah, Eman S H; Badary, Dalia M

    2017-03-01

    Folic acid plays an important role in cellular metabolic activities. The present study was designed to investigate the protective effect of folic acid against lead acetate-induced hepatotoxicity. Twenty four male Wistar albino rats were randomly divided into four groups, six animals each. Negative control group received the vehicle, positive control group received 1mg/kg folic acid for five consecutive days/week for 4 weeks orally, lead-exposed group received 10mg/kg lead acetate intraperitoneally (IP) for five consecutive days/week for 4 weeks, and lead-treated group received 10mg/kg lead acetate IP and 1mg/kg folic acid orally for five consecutive days/week for 4 weeks concurrently. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ- glutamyltransferase (GGT) were measured. Hepatic total peroxide and interleukin-1β (IL-1β) were also investigated. Histopathological studies using hematoxylin-eosin (H&E) and periodic acid shiff's (PAS) were carried out. The expression of nuclear factor kappa B (NF-κB) was evaluated using immunohistochemistry. Serum AST, ALT and GGT and hepatic total peroxide and IL-1β were significantly increased in lead-exposed group and were positively correlated with hepatic lead level. Moreover, lead-exposed rats showed hydropic degeneration, nuclear vesiculation, high lymphocytic infiltration, depletion of glycogen content and NF-κB expression. Concomitant folic acid administration resulted in a significant alleviation of biochemical and structural alteration-induced by lead. This was associated with reduction of hepatic total peroxide and IL-1β and reduction of NF-κB expression. In conclusion, folic acid protects against lead acetate-induced hepatotoxicity by decreasing NF-κB, IL-1β production and lipid peroxidation mediataed cell injury.

  2. Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells.

    PubMed

    Wu, Pei-Yu; Lin, Yu-Chia; Chang, Chia-Ling; Lu, Hsing-Tsen; Chin, Chia-Hsuan; Hsu, Tsan-Ting; Chu, Dachen; Sun, Synthia H

    2009-06-01

    Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined.We characterized the role of P2X7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X7 receptors. Functional inhibition of P2X7 receptors by P2X7 receptor selective antagonists, 5'-triphosphate, periodate-oxidized 2',3'-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca2+ concentrations ([Ca2+]i) were decreased in either RA- or oATP-differentiated or P2X7receptor knockdown N2a cells. Simply cultured N2a cells in low Ca2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X7 receptors are involved in neuronal differentiation.We provide additional evidence shown that the ATP release-activated P2X7 receptor is important in maintaining cell survival of N2a neuroblastoma cells.

  3. Effect of dietary fibers on cholic acid induced cell proliferation in the colonic epithelium of C57BL/6J mice

    SciTech Connect

    Robblee, N.M.; Bruce, W.R.; Bird, R.P.

    1986-03-01

    It has been postulated that high fat diets promote tumorigenesis by increasing the level of secondary bile acids in the colonic lumen. Dietary fibers are thought to be protective perhaps through their interaction with bile acids. In the present study, animals were fed diets containing either 0%, 5%, or 10% cellulose (C), pectin (P), or wheat bran (WB). The diets were formulated to contain either 0% (control) or 0.2% cholic acid (test). After two weeks of dietary treatment the animals were injected with (/sup 3/H)-thymidine and their colons were processed for autoradiography. The number of labeled cells (LC) in the colonic crypts was determined. Among the control diets, 10%P induced a two-fold increase in the LC. All the test groups had significantly higher LC than in their controls. However, the C group excited a higher LC than the P or WB groups (5.2 +/- 0.8 vs 3.9 +/- 0.8 or 3.9 +/- 0.6). These results were substantiated by metaphase arrest technique. The authors results show that nonfermentable fiber does not alleviate bile acid induced cell proliferative activity in the colon whereas fermentable fibers will counteract the promotional effect of a high fat diet.

  4. Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP.

    PubMed

    Chao, C C; Sun, N K; Lin-Chao, S

    1993-02-15

    A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

  5. Effects of Histone Deacetylase Inhibitor (Valproic Acid) on the Expression of Hypoxia-inducible Factor-1 Alpha in Human Retinal Müller Cells

    PubMed Central

    Kim, Young Jun; Park, Sang Jun; Kim, Na Rae

    2017-01-01

    Purpose To evaluate the effects of valproic acid (VPA), a histone deacetylase inhibitor (HDACI), on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in human retinal Müller cells under hypoxic conditions. Methods Chemical hypoxia was induced in human retinal Müller cells (MIO-M1) by treatment with increasing concentrations of cobalt(II) chloride (CoCl2). Müller cells were also treated with a set concentration of CoCl2, along with various concentrations of VPA. The expression of HIF-1α and VEGF in the treated Müller cells was determined by enzyme-linked immunosorbent assay. Results Exposure of human retinal Müller cells to increasing concentrations of CoCl2 produced a dose-dependent increase in HIF-1α expression. The addition of increasing concentrations of VPA lead to a dose-dependent decrease in expression of HIF-1α and VEGF in Müller cells exposed to a set concentration of CoCl2. Conclusions HDACI VPA downregulated the expressions of HIF-1α and VEGF in human retinal Müller cells under hypoxic conditions. Using HDACI to target HIF-1α expression in Müller cells could be a new therapeutic strategy for the treatment of retinal vascular diseases. PMID:28243027

  6. Increased beta-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation.

    PubMed

    Perdomo, German; Commerford, S Renee; Richard, Ann-Marie T; Adams, Sean H; Corkey, Barbara E; O'Doherty, Robert M; Brown, Nicholas F

    2004-06-25

    Skeletal muscle insulin resistance may be aggravated by intramyocellular accumulation of fatty acid-derived metabolites that inhibit insulin signaling. We tested the hypothesis that enhanced fatty acid oxidation in myocytes should protect against fatty acid-induced insulin resistance by limiting lipid accumulation. L6 myotubes were transduced with adenoviruses encoding carnitine palmitoyltransferase I (CPT I) isoforms or beta-galactosidase (control). Two to 3-fold overexpression of L-CPT I, the endogenous isoform in L6 cells, proportionally increased oxidation of the long-chain fatty acids palmitate and oleate and increased insulin stimulation of [(14)C]glucose incorporation into glycogen by 60% while enhancing insulin-stimulated phosphorylation of p38MAPK. Incubation of control cells with 0.2 mm palmitate for 18 h caused accumulation of triacylglycerol, diacylglycerol, and ceramide (but not long-chain acyl-CoA) and decreased insulin-stimulated [(14)C]glucose incorporation into glycogen (60%), [(3)H]deoxyglucose uptake (60%), and protein kinase B phosphorylation (20%). In the context of L-CPT I overexpression, palmitate preincubation produced a relative decrease in insulin-stimulated incorporation of [(14)C]glucose into glycogen (60%) and [(3)H]deoxyglucose uptake (40%) but did not inhibit phosphorylation of protein kinase B. Due to the enhancement of insulin-stimulated glucose metabolism induced by L-CPT I overexpression itself, net insulin-stimulated incorporation of [(14)C]glucose into glycogen and [(3)H]deoxyglucose uptake in L-CPT I-transduced, palmitate-treated cells were significantly greater than in palmitate-treated control cells (71 and 75% greater, respectively). However, L-CPT I overexpression failed to decrease intracellular triacylglycerol, diacylglycerol, ceramide, or long-chain acyl-CoA. We propose that accelerated beta-oxidation in muscle cells exerts an insulin-sensitizing effect independently of changes in intracellular lipid content.

  7. Immunohistochemical characterization of glial fibrillary acidic protein (GFAP)-expressing cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

    PubMed

    Tennakoon, Anusha Hemamali; Izawa, Takeshi; Wijesundera, Kavindra Kumara; Murakami, Hiroshi; Katou-Ichikawa, Chisa; Tanaka, Miyuu; Golbar, Hossain M; Kuwamura, Mitsuru; Yamate, Jyoji

    2015-01-01

    Hepatic stellate cells, the principal fibrogenic cell type in the liver, are known to express the astrocyte marker glial fibrillary acidic protein (GFAP). However, the exact role of GFAP-expressing cells in liver fibrosis remains to be elucidated. In this study, cellular properties of GFAP-expressing cells were investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100 mg/kg BW, twice a week) and examined at post first injection weeks 5, 10, 15, 20 and 25. Appearance of GFAP-expressing myofibroblasts peaked at week 15, associated with fibrosis progression. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and alpha-smooth muscle actin. Some GFAP-positive myofibroblasts co-expressed nestin (neural stem cell marker), while a few co-expressed A3 (mesenchymal stem cell marker) and Thy-1 (immature mesenchymal cell marker). A few GFAP expressing cells underwent both mitosis and apoptosis. These results indicate that there is a dynamic participation of GFAP-expressing myofibroblasts in rat liver cirrhosis, and that they are mainly derived from hepatic stellate cells, and partly from cells in the stem cell lineage. These findings, which were shown for the first time in detail, would be useful to understand the role of GFAP-expressing myofibroblasts in the pathogenesis of chemically induced liver cirrhosis.

  8. All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

    PubMed

    Mangiarotti, R; Danova, M; Alberici, R; Pellicciari, C

    1998-01-01

    In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.

  9. All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

    PubMed Central

    Mangiarotti, R.; Danova, M.; Alberici, R.; Pellicciari, C.

    1998-01-01

    In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity. PMID:9460987

  10. Heat shock of cultured GC cells enhances the level of triiodothyronine induced growth hormone (GH) and GH messenger ribonucleic acid.

    PubMed

    Shapiro, L E; Katz, C P; DeFesi, C R; Surks, M I

    1989-07-01

    We have previously proposed that the effects of heat shock on thyroid hormone-responsive rat pituitary tumor (GC) cells may be a model relevant to the in vivo effects of nonthyroidal disease on thyroid hormone action. To determine the effects of heat shock on thyroid hormone responses, GC cells (normally cultured at 37 C) were studied after incubation at 41 C. After 18 h at 41 C there was enhanced synthesis of proteins (mol wt, 70,000 and 90,000) considered to be universal markers of the cellular response to heat shock. Incubation at 41 C also resulted in a significant decrease in GC cell viability and (after 24 h) arrest of GC cell growth. However, the induction of GH synthesis by T3 was significantly enhanced in GC cells stressed by incubation at 41 C. The addition of 5 nM T3 to thyroid hormone-depeleted GC cells resulted in a significantly greater (P less than 0.001) accumulation of GH (2642 +/- 280 ng/18 h) during 41 C incubation than during 37 C incubation (1223 +/- 175 ng/18 h). The enhanced T3-induced production of GH was coincident with a proportional increase (P less than 0.05) in cellular GH mRNA determined by dot hybridization analysis. Thus, the stress of 41 C incubation elicits a heat shock response in GC cells characterized by decreased viability and growth arrest, but enhanced accumulation of GH mRNA in response to T3. Our recent report on the identical effects due to the stress of implantation of the Walker 256 carcinoma on T3-induced rat pituitary GH mRNA in vivo suggests that heat shock of cultured GC cells is a valid in vitro model of nonthyroidal disease.

  11. 3,4-Dihydroxyphenylacetic acid, a microbiota-derived metabolite of quercetin, protects against pancreatic β-cells dysfunction induced by high cholesterol.

    PubMed

    Carrasco-Pozo, Catalina; Gotteland, Martin; Castillo, Rodrigo L; Chen, Chen

    2015-06-10

    Cholesterol plays an important role in inducing pancreatic β-cell dysfunction, characterized by an impaired insulin secretory response to glucose, representing a hallmark of the transition from pre-diabetes to diabetes. 3,4 dihydroxyphenylacetic acid (ES) is a scarcely studied microbiota-derived metabolite of quercetin with antioxidant properties. The aim of this study was to determine the protective effect of ES against apoptosis, mitochondrial dysfunction and oxidative stress induced by cholesterol in Min6 pancreatic β-cells. Cholesterol decreased viability, induced apoptosis and mitochondrial dysfunction by reducing complex I activity, mitochondrial membrane potential, ATP levels and oxygen consumption. Cholesterol promoted oxidative stress by increasing cellular and mitochondrial reactive oxygen species and lipid peroxidation and decreasing antioxidant enzyme activities; in addition, it slightly increased Nrf2 translocation to the nucleus. These events resulted in the impairment of the glucose-induced insulin secretion. ES increased Nrf2 translocation to the nucleus and protected pancreatic β-cells against impaired insulin secretion induced by cholesterol by preventing oxidative stress, apoptosis and mitochondrial dysfunction. Nrf2 activation seems to be involved in the mechanisms underlying the antioxidant protection exerted by ES in addition to preventing the disruption of antioxidant enzymatic defenses. Although additional in vivo experiments are required, this metabolite is suggested as a promising drug target for the prevention of the pathological development from a pre-diabetic to a diabetic state.

  12. Regulatory CD8{sup +} T cells induced by exposure to all-trans retinoic acid and TGF-{beta} suppress autoimmune diabetes

    SciTech Connect

    Kishi, Minoru; Yasuda, Hisafumi; Abe, Yasuhisa; Sasaki, Hirotomo; Shimizu, Mami; Arai, Takashi; Okumachi, Yasuyo; Moriyama, Hiroaki; Hara, Kenta; Yokono, Koichi; Nagata, Masao

    2010-03-26

    Antigen-specific regulatory CD4{sup +} T cells have been described but there are few reports on regulatory CD8{sup +} T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8{sup +} T cells from 8.3-NOD transgenic mice. CD8{sup +} T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-{beta}, and all-trans retinoic acid (ATRA) for 5 days. CD8{sup +} T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-{beta} and ATRA had low Foxp3{sup +} expression (1.7 {+-} 0.9% and 3.2 {+-} 4.5%, respectively). In contrast, CD8{sup +} T cells induced by exposure to IGRP, SpDCs, TGF-{beta}, and ATRA showed the highest expression of Foxp3{sup +} in IGRP-reactive CD8{sup +} T cells (36.1 {+-} 10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8{sup +} T cells cultured with IGRP, SpDCs, TGF-{beta}, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8{sup +} T cells suppressed the proliferation of diabetogenic CD8{sup +} T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-{beta} induces CD8{sup +}Foxp3{sup +} T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.

  13. Disruption of BSEP Function in HepaRG Cells Alters Bile Acid Disposition and Is a Susceptive Factor to Drug-Induced Cholestatic Injury.

    PubMed

    Qiu, Xi; Zhang, Yueping; Liu, Tongtong; Shen, Hong; Xiao, Yongling; Bourner, Maureen J; Pratt, Jennifer R; Thompson, David C; Marathe, Punit; Humphreys, W Griffith; Lai, Yurong

    2016-04-04

    In the present study, we characterized in vitro biosynthesis and disposition of bile acids (BAs) as well as hepatic transporter expression followed by ABCB11 (BSEP) gene knockout in HepaRG cells (HepaRG-KO cells). BSEP KO in HepaRG cells led to time-dependent BA accumulation, resulting in reduced biosynthesis of BAs and altered BA disposition. In HepaRG-KO cells, the expression of NTCP, OATP1B1, OATP2B1, BCRP, P-gp, and MRP2 were reduced, whereas MRP3 and OCT1 were up-regulated. As a result, BSEP KO altered the disposition of BAs and subsequently underwent adaptive regulations of BA synthesis and homeostasis to enable healthy growth of the cells. Although BSEP inhibitors caused no or slight increase of BAs in HepaRG wild type cells (HepaRG-WT cells), excessive intracellular accumulation of BAs was observed in HepaRG-KO cells exposed to bosentan and troglitazone, but not dipyridamole. LDH release in the medium was remarkably increased in HepaRG-KO cultures exposed to troglitazone (50 μM), suggesting drug-induced cellular injury. The results revealed that functional impairment of BSEP predisposes the cells to altered BA disposition and is a susceptive factor to drug-induced cholestatic injury. In total, BSEP inhibition might trigger the processes but is not a sole determinant of cholestatic cellular injury. As intracellular BA accumulation is determined by BSEP function and the subsequent adaptive gene regulation, assessment of intracellular BA accumulation in HepaRG-KO cells could be a useful approach to evaluate drug-induced liver injury (DILI) potentials of drugs that could disrupt other BA homeostasis pathways beyond BSEP inhibition.

  14. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

    SciTech Connect

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-08

    Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

  15. Salvia miltiorrhiza water-soluble extract, but not its constituent salvianolic acid B, abrogates LPS-induced NF-κB signalling in intestinal epithelial cells

    PubMed Central

    Kim, J S; Narula, A S; Jobin, C

    2005-01-01

    Herbal medicine has become an increasing popular therapeutic alternative among patients suffering from various inflammatory disorders. The Salvia miltiorrhizae water-soluble extract (SME) have been shown to possess antioxidant and anti-inflammatory properties in vitro. However, the mechanism of action and impact of SME on LPS-induced gene expression is still unknown. We report that SME significantly abrogated LPS-induced IκB phosphorylation/degradation, NF-κB transcriptional activity and ICAM-1 gene expression in rat IEC-18 cells. Chromatin immunoprecipitation assay demonstrated that LPS-induced RelA recruitment to the ICAM-1 gene promoter was inhibited by SME. Moreover, in vitro kinase assay showed that SME directly inhibits LPS induced IκB kinase (IKK) activity in IEC-18 cells. To investigate the physiological relevance of SME inhibitory activity on NF-κB signalling, we used small intestinal explants and primary intestinal epithelial cells derived from a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-κB cis-elements (cis-NF-κBEGFP). SME significantly blocked LPS-induced EGFP expression and IκBα phosphorylation in intestinal explants and primary IECs, respectively. However, salvianolic acid B, an activate component of SME did not inhibit NF-κB transcriptional activity and IκB phosphorylation/degradation in IEC-18 cells. These results indicate that SME blocks LPS-induced NF-κB signalling pathway by targeting the IKK complex in intestinal epithelial cells. Modulation of bacterial product-mediated NF-κB signalling by natural plant extracts may represent an attractive strategy towards the prevention and treatment of intestinal inflammation. PMID:15996193

  16. Salvia miltiorrhiza water-soluble extract, but not its constituent salvianolic acid B, abrogates LPS-induced NF-kappaB signalling in intestinal epithelial cells.

    PubMed

    Kim, J S; Narula, A S; Jobin, C

    2005-08-01

    Herbal medicine has become an increasing popular therapeutic alternative among patients suffering from various inflammatory disorders. The Salvia miltiorrhizae water-soluble extract (SME) have been shown to possess antioxidant and anti-inflammatory properties in vitro. However, the mechanism of action and impact of SME on LPS-induced gene expression is still unknown. We report that SME significantly abrogated LPS-induced IkappaB phosphorylation/degradation, NF-kappaB transcriptional activity and ICAM-1 gene expression in rat IEC-18 cells. Chromatin immunoprecipitation assay demonstrated that LPS-induced RelA recruitment to the ICAM-1 gene promoter was inhibited by SME. Moreover, in vitro kinase assay showed that SME directly inhibits LPS induced IkappaB kinase (IKK) activity in IEC-18 cells. To investigate the physiological relevance of SME inhibitory activity on NF-kappaB signalling, we used small intestinal explants and primary intestinal epithelial cells derived from a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-kappaB cis-elements (cis-NF-kappaB(EGFP)). SME significantly blocked LPS-induced EGFP expression and IkappaBalpha phosphorylation in intestinal explants and primary IECs, respectively. However, salvianolic acid B, an activate component of SME did not inhibit NF-kappaB transcriptional activity and IkappaB phosphorylation/degradation in IEC-18 cells. These results indicate that SME blocks LPS-induced NF-kappaB signalling pathway by targeting the IKK complex in intestinal epithelial cells. Modulation of bacterial product-mediated NF-kappaB signalling by natural plant extracts may represent an attractive strategy towards the prevention and treatment of intestinal inflammation.

  17. Mitochondrial genome depletion dysregulates bile acid- and paracetamol-induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells

    PubMed Central

    Perez, MJ; Gonzalez-Sanchez, E; Gonzalez-Loyola, A; Gonzalez-Buitrago, JM; Marin, JJG

    2011-01-01

    BACKGROUND AND PURPOSE Mitochondria are involved in the toxicity of several compounds, retro-control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated. EXPERIMENTAL APPROACH Hepa 1-6 mouse hepatoma cells with 70% decrease in 16S/18S rRNA ratio (Rho cells) were obtained by long-term treatment with ethidium bromide. KEY RESULTS Spontaneous apoptosis and reactive oxygen species (ROS) generation were decreased in Rho cells. Following glycochenodeoxycholic acid (GCDCA) or paracetamol, Rho cells generated less ROS and were more resistant to cell death. Apoptosis induced by GCDCA and Fas was also reduced. The basal expression of Mdr1 was significantly enhanced, but this was not further stimulated by GCDCA or paracetamol, as observed in wild-type (WT) cells. Basal expression of Mrp1 and Mrp4 was similar in WT and Rho cells, whereas they were up-regulated only in WT cells after GCDCA or paracetamol, along with the transcription factors Shp and Nrf2, but not Fxr or Pxr. Increased expression of Nrf2 was accompanied by its enhanced nuclear translocation. Glycoursodeoxycholic acid failed to cause any of the effects observed for GCDCA or paracetamol. CONCLUSIONS AND IMPLICATIONS The Nrf2-mediated pathway is partly independent of ROS production. Nuclear translocation of Nrf2 is insufficient to up-regulate Mdr1, Mrp1 and Mrp4, which requires the participation of other regulatory element(s) whose activation in response to GCDCA and paracetamol is impaired in Rho cells and hence probably sensitive to ROS. PMID:21175587

  18. Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells

    PubMed Central

    Huang, Hao; Hong, Qian; Tan, Hong-ling; Xiao, Cheng-rong; Gao, Yue

    2016-01-01

    Aim: Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells. Methods: PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA. Results: Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10–40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2. Conclusion: The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS-induced

  19. JA, a new type of polyunsaturated fatty acid isolated from Juglans mandshurica Maxim, limits the survival and induces apoptosis of heptocarcinoma cells.

    PubMed

    Gao, Xiu-Li; Lin, Hua; Zhao, Wei; Hou, Ya-Qin; Bao, Yong-Li; Song, Zhen-Bo; Sun, Lu-Guo; Tian, Shang-Yi; Liu, Biao; Li, Yu-Xin

    2016-03-01

    Juglans mandshurica Maxim (Juglandaceae) is a famous folk medicine for cancer treatment and some natural compounds isolated from it have been studied extensively. Previously we isolated a type of ω-9 polyunsaturated fatty acid (JA) from the bark of J. mandshurica, however little is known about its activity and the underlying mechanisms. In this study, we studied anti-tumor activity of JA on several human cancer cell lines. Results showed that JA is cytotoxic to HepG2, MDA-MB-231, SGC-7901, A549 and Huh7 cells at a concentration exerting minimal toxic effects on L02 cells. The selective toxicity of JA was better than other classical anti-cancer drugs. Further investigation indicated that JA could induce cell apoptosis, characterized by chromatin condensation, DNA fragmentation and activation of the apoptosis-associated proteins such as Caspase-3 and PARP-1. Moreover, we investigated the cellular apoptosis pathway involved in the apoptosis process in HepG2 cells. We found that proteins involved in mitochondrion (cleaved-Caspase-9, Apaf-1, HtrA2/Omi, Bax, and Mitochondrial Bax) and endocytoplasmic reticulum (XBP-1s, GRP78, cleaved-Caspase-7 and cleaved-Caspase-12) apoptotic pathways were up-regulated when cells were treated by JA. In addition, a morphological change in the mitochondrion was detected. Furthermore, we found that JA could inhibit DNA synthesis and induce G2/M cell cycle arrest. The expression of G2-to-M transition related proteins, such as CyclinB1 and phosphorylated-CDK1, were reduced. In contrast, the G2-to-M inhibitor p21 was increased in JA-treated cells. Overall, our results suggest that JA can induce mitochondrion- and endocytoplasmic reticulum-mediated apoptosis, and G2/M phase arrest in HepG2 cells, making it a promising therapeutic agent against hepatoma.

  20. Ultrasound-potentiated salicylic acid-induced physiological effects and production of taxol in hazelnut (Corylus avellana L.) cell culture.

    PubMed

    Rezaei, Ayatollah; Ghanati, Faezeh; Behmanesh, Mehrdad; Mokhtari-Dizaji, Manijhe

    2011-11-01

    Effects of ultrasound (US), salicylic acid (SA) and their combined use on the growth and secondary metabolite production of suspension-cultured Corylus avellana cells were investigated. The cultures were treated with US (40 kHz) for short periods of time (2, 3, 5 and 10 min) and SA (25 and 50 mg L(-1)). Results showed that although phenolic content of the cells was significantly increased under exposure to treatments, flavonoids content significantly decreased. Taxol biosynthesis was improved by all treatments. US exposure increased the extracellular, cell-associated and total taxol yield three-, 1.6-, and two-fold compared with that of the control, respectively. SA at all levels was more effective than US in stimulating cell-associated and total taxol production. Combined treatment of US and SA at 50 mg L(-1) resulted in the most improvement in total taxol production, which was about seven times higher than that of the US, three times higher than that of the SA and 14 times higher than that of the control. The results suggest a synergism between US and SA in enhancing taxol production by hazelnut cells.

  1. Deoxycholic acid inhibited proliferation and induced apoptosis and necrosis by regulating the activity of transcription factors in rat pancreatic acinar cell line AR42J.

    PubMed

    Zhang, Guixin; Zhang, Jingwen; Shang, Dong; Qi, Bing; Chen, Hailong

    2015-09-01

    The objective of this study is to investigate the effect of deoxycholic acid (DCA) on rat pancreatic acinar cell line AR42J and the functional mechanisms of DCA on AR42J cells. AR42J cells were treated with various concentrations of DCA for 24 h and also treated with 0.4 mmol/L DCA for multiple times, and then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect the AR42J cell survival rate. Flow cytometric was used to detect the cell apoptosis and necrosis in AR42J cells treated with 0.4 mmol/L and 0.8 mmol/L DCA. The cells treated with phosphate buffer saline (PBS) were served as control. In addition, the DNA-binding activity assays of transcription factors (TFs) in nuclear proteins of cells treated with DCA were determined using Panomics Procarta Transcription Factor Assay Kit. The relative survival rates were markedly decreased (P < 0.05) in a dose- and time-dependent manner. Compared with control group, the cell apoptosis and necrosis ratio were both significantly elevated in 0.4 mmol/L DCA and 0.8 mmol/L DCA groups (P < 0.01). A significant increase (P < 0.05) in the activity of transcription factor 2 (ATF2), interferon-stimulated response element (ISRE), NKX-2.5, androgen receptor (AR), p53, and hypoxia-inducible factor-1 (HIF-1) was observed, and the activity of peroxisome proliferator-activated receptor (PPAR), activator protein 1 (AP1), and E2F1 was reduced (P < 0.05). In conclusion, DCA inhibited proliferation and induced apoptosis and necrosis in AR42J cells. The expression changes of related genes regulated by TFs might be the molecular mechanism of AR42J cell injury.

  2. Effect of Vitamin E and Omega-3 Fatty Acids on Protecting Ambient PM2.5-Induced Inflammatory Response and Oxidative Stress in Vascular Endothelial Cells

    PubMed Central

    Bo, Liang; Jiang, Shuo; Xie, Yuquan; Kan, Haidong; Song, Weimin; Zhao, Jinzhuo

    2016-01-01

    Although the mechanisms linking cardiopulmonary diseases to ambient fine particles (PM2.5) are still unclear, inflammation and oxidative stress play important roles in PM2.5-induced injury. It is well known that inflammation and oxidative stress could be restricted by vitamin E (Ve) or omega-3 fatty acids (Ω-3 FA) consumption. This study investigated the effects of Ve and Ω-3 FA on PM2.5-induced inflammation and oxidative stress in vascular endothelial cells. The underlying mechanisms linking PM2.5 to vascular endothelial injury were also explored. Human umbilical vein endothelial cells (HUVECs) were treated with 50 μg/mL PM2.5 in the presence or absence of different concentrations of Ve and Ω-3 FA. The inflammatory cytokines and oxidative stress markers were determined. The results showed that Ve induced a significant decrease in PM2.5-induced inflammation and oxidative stress. Malondialdehyde (MDA) in supernatant and reactive oxygen species (ROS) in cytoplasm decreased by Ve, while the superoxide dismutase (SOD) activity elevated. The inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) also reduced by Ve. Moreover, Ω-3 FA played the same role on decreasing the inflammation and oxidative stress. IL-6 and TNF-α expressions were significantly lower in combined Ve with Ω-3 FA than treatment with Ve or Ω-3 FA alone. The Ve and Ω-3 FA intervention might abolish the PM2.5-induced oxidative stress and inflammation in vascular endothelial cells. There might be an additive effect of these two nutrients in mediating the PM2.5-induced injury in vascular endothelial cells. The results suggested that inflammation and oxidative stress might be parts of the mechanisms linking PM2.5 to vascular endothelial injury. PMID:27007186

  3. Quercetin and Ascorbic Acid Suppress Fructose-Induced NLRP3 Inflammasome Activation by Blocking Intracellular Shuttling of TXNIP in Human Macrophage Cell Lines.

    PubMed

    Choe, Jung-Yoon; Kim, Seong-Kyu

    2017-03-22

    The aim of this study was to identify the role of thioredoxin-interacting protein (TXNIP) and its interaction with antioxidants in the activation of the fructose-induced NOD-like receptor protein 3 (NLRP3) inflammasome in human macrophages. The study was performed with U937 and THP-1 macrophage cell lines. Total reactive oxygen species (ROS) were measured by flow cytometry. Interleukin-1β (IL-1β), IL-18, NLRP3, TXNIP, and caspase-1 protein expression was detected using western blotting. Quantitative real-time polymerase chain reaction was used to detect IL-1β, IL-18, and caspase-1 gene expression. Intracellular shuttling of TXNIP was assessed by immunofluorescent staining with MitoTracker Red. Increased production of ROS and expression of IL-1β, IL-18, and caspase-1 genes and proteins were observed in U937 and THP-1 cells incubated with fructose and were effectively inhibited by quercetin and ascorbic acid. Intracellular shuttling of TXNIP from the nucleus into the mitochondria was detected under stimulation with fructose, which was also attenuated by antioxidants quercetin and ascorbic acid but not butylated hydroxyanisole. Treatment of macrophages with fructose promoted the association between TXNIP and NLRP3 in the cytosol, sequentially resulting in the activation of the NLRP3 inflammasome. This study revealed that intracellular TXNIP protein is a critical regulator of activation of the fructose-induced NLRP3 inflammasome, which can be effectively blocked by the antioxidants quercetin and ascorbic acid.

  4. Caffeoylquinic Acid Derivatives Protect SH-SY5Y Neuroblastoma Cells from Hydrogen Peroxide-Induced Injury Through Modulating Oxidative Status.

    PubMed

    Jiang, Xiao-Wen; Bai, Jun-Peng; Zhang, Qiao; Hu, Xiao-Long; Tian, Xing; Zhu, Jun; Liu, Jia; Meng, Wei-Hong; Zhao, Qing-Chun

    2017-04-01

    Oxidative stress has been confirmed as a contribution to the pathogenesis and pathophysiology of many neurological disorders such as Alzheimer's disease and Parkinson's disease. Caffeoylquinic acids (CQAs) are considered to have anti-oxidative stress ability in a previous study, but the structure-activity relationships (SARs) of CQAs in neuroprotective effects are still unclear. In the present study, we primarily expound the SARs of CQAs in counteracting H2O2-induced injury in SH-SY5Y cells. We found that CQAs (1-10) represented the protection of SH-SY5Y cells against H2O2-induced injury in varying degrees and malonyl groups could obviously increase the anti-oxidative stress ability of CQAs. Intensive studies of 4,5-O-dicaffeoyl-1-O-(malic acid methyl ester)-quinic acid (MDCQA) indicated that the mechanisms could potentially involve activation of endogenous antioxidant enzymes and the regulation of the phosphorylation of MAPKs and AKT. In conclusion, MDCQA could serve as a neuroprotective agent with a potential to attenuate oxidative stress.

  5. Chebulagic acid inhibits the LPS-induced expression of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation.

    PubMed

    Liu, Yueying; Bao, Luer; Xuan, Liying; Song, Baohua; Lin, Lin; Han, Hao

    2015-07-01

    Inflammatory response in the vasculature, including the overexpression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, has been demonstrated to increase the risk of thrombosis development. Chebulagic acid (CA) is a key chemical component in the traditional Mongolian anti-thrombotic drug Garidi-13, and has been suggested to exert anti-inflammatory and anti-infective effects. The present study aimed to evaluate the regulatory impact of CA on a number of biological processes, including lipopolysaccharide (LPS)-induced inflammation, LPS-promoted mitogen-activated protein kinase (MAPK) activation and the expression of toll-like receptor (TLR)4 in EA.hy926 human endothelial cells. The results indicated that CA significantly inhibited the LPS-induced upregulation of TNF-α and IL-1β in a dose- and time-dependent manner. Furthermore, LPS-activated MAPK signaling was inhibited by CA treatment in the EA.hy926 cells. However, TLR4, which serves a key function in LPS-induced inflammation as the receptor of LPS, was not regulated by the CA treatment. In summary, the results of the present study indicate that CA inhibits the LPS-induced promotion of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation, which may contribute to the anti-thrombotic effect of Garidi-13.

  6. Sargaquinoic Acid Inhibits TNF-α-Induced NF-κB Signaling, Thereby Contributing to Decreased Monocyte Adhesion to Human Umbilical Vein Endothelial Cells (HUVECs).

    PubMed

    Gwon, Wi-Gyeong; Lee, Bonggi; Joung, Eun-Ji; Choi, Min-Woo; Yoon, Nayoung; Shin, Taisun; Oh, Chul-Woong; Kim, Hyeung-Rak

    2015-10-21

    Sargaquinoic acid (SQA) has been known for its antioxidant and anti-inflammatory properties. This study investigated the effects of SQA isolated from Sargassum serratifolium on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). SQA decreased the expression of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as chemotactic cytokines such as interleukin-8 and monocyte chemoattractant protein-1 in TNF-α-treated HUVECs. As a result, SQA prevented monocyte adhesion to TNF-α-induced adhesion. SQA also inhibited TNF-α-induced nuclear factor kappa B (NF-κB) translocation into the nucleus by preventing proteolytic degradation of inhibitor κB-α. Overall, SQA protects against TNF-α-induced vascular inflammation through inhibition of the NF-κB pathway in HUVECs. These data suggest that SQA may be used as a therapeutic agent for vascular inflammatory diseases such as atherosclerosis.

  7. Trans-10, cis-12 conjugated linoleic acid and the PPAR-γ agonist rosiglitazone attenuate lipopolysaccharide-induced TNF-α production by bovine immune cells.

    PubMed

    Perdomo, M C; Santos, J E; Badinga, L

    2011-10-01

    Lipopolysaccharide (LPS) modulates innate immunity through alteration of cytokine production by immune cells. The objective of this study was to examine the effect of exogenous conjugated linoleic acid (CLA) and PPAR-γ agonist, rosiglitazone, on LPS-induced tumor necrosis factor α (TNF-α) production by cultured whole blood from prepubertal Holstein heifers (mean age, 5.5 mo). Compared with unstimulated cells, addition of LPS (10 μg/mL) to the culture medium increased (P<0.03) peripheral blood mononuclear cell proliferation≤2.5-fold. Coincubation with interferon γ (5 ng/mL) further stimulated (P<0.01) the lymphoproliferative response to LPS. Lipopolysaccharide increased (P<0.01) TNF-α concentration in cultured whole blood in a dose- and time-dependent manner. The greatest TNF-α stimulation occurred after 12 h of exposure to 1 μg/mL LPS. Coincubation with trans-10, cis-12 CLA isomer (100 μM) or rosiglitazone (10 μM), a PPAR-γ agonist, decreased (P<0.01) LPS-induced TNF-α production by 13% and 29%, respectively. Linoleic acid and cis-9, trans-11 CLA isomer had no detectable effects on LPS-induced TNF-α production in cultured bovine blood. The PPAR-γ agonist-induced TNF-α attenuation was reversed when blood was treated with both rosiglitazone and GW9662, a selective PPAR-γ antagonist. Addition of rosiglitazone to the culture medium tended to reduce nuclear factor-κ Bp65 concentration in nuclear and cytosolic extracts isolated from cultured peripheral blood mononuclear cells. Results show that LPS is a potent inducer of TNF-α production in bovine blood cells and that trans-10, cis-12 CLA and PPAR-γ agonists may attenuate the pro-inflammatory response induced by LPS in growing dairy heifers. Additional studies are needed to fully characterize the involvement of nuclear factor-κ B in LPS signaling in bovine blood cells.

  8. Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells.

    PubMed

    Lee, Yean-Jang; Kuo, Hsing-Chun; Chu, Chia-Yih; Wang, Chau-Jong; Lin, Wan-Chyi; Tseng, Tsui-Hwa

    2003-12-15

    Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological and pharmacological activities including antioxidant, anti-inflammation, antiviral action, and anticancer effect. Our previous studies showed that CAPE exhibited significant cytotoxicity in oral cancer cells. Herein we further investigated the cytotoxicity potential of CAPE and the mechanism of its action in C6 glioma cells. The data exhibited that C6 glioma cells underwent internucleosomal DNA fragmentation 24 hr after the treatment of CAPE (50 microM). The proportion of C6 glioma cells with hypodiploid nuclei was increased to 24% at 36 hr after the exposure. Further results showed that CAPE induced the release of cytochrome c from mitochondria into cytosol, and the activation of CPP32. CAPE application also enhanced the expression of p53, Bax, and Bak. Finally, the potential signaling components underlying CAPE induction of apoptosis were elucidated. We found that CAPE activated extracellular signal-regulated kinase (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in C6 glioma cells. More importantly, p38 kinase formed a complex with p53 after the treatment of CAPE for 0.5 hr. The expression of p53, phospho-serine 15 of p53, and Bax, and inactivate form of CPP32 was suppressed by a pretreatment of a specific p38 MAPK inhibitor, SB203580. The resultant data suggest that p38 MAPK mediated the CAPE-induced p53-dependent apoptosis in C6 glioma cells.

  9. Disturbed apoptosis and cell proliferation in developing neuroepithelium of lumbo-sacral neural tubes in retinoic acid-induced spina bifida aperta in rat.

    PubMed

    Wei, Xiaowei; Li, Hui; Miao, Jianing; Zhou, Fenghua; Liu, Bo; Wu, Di; Li, Shujing; Wang, Lili; Fan, Yang; Wang, Weilin; Yuan, Zhengwei

    2012-08-01

    Spina bifida is a complex congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. However, the cellular mechanism underlying spina bifida is not fully understood. Here, we investigated cell apoptosis in whole embryos and proliferation of neural progenitor cells in the spinal neural tube during neurulation in all-trans retinoic acid (atRA)-induced spina bifida in fetal rats. Cell apoptosis was assessed by TUNEL assay on whole-mount and serially sectioned samples of rat embryos with spina bifida. Cell proliferation of lumbo-sacral neural progenitor cells was assessed by staining for the mitotic marker Ki67 and pH3. We found an excess of apoptosis in the neuroepithelium of embryos with spina bifida, which became more marked as embryos progress from E11 to E13. Conversely, there was a reduction in cell proliferation in spina bifida embryos, with a progressively greater difference from controls with stage from E11 to 13. Thus, atRA-induced spina bifida in rat shows perturbed apoptosis and proliferation of neural progenitors in the lumbo-sacral spinal cord during embryonic development, which might contribute to the pathogenesis of spina bifida.

  10. The Vitamin A Derivative All-Trans Retinoic Acid Repairs Amyloid-β-Induced Double-Strand Breaks in Neural Cells and in the Murine Neocortex.

    PubMed

    Gruz-Gibelli, Emmanuelle; Chessel, Natacha; Allioux, Clélia; Marin, Pascale; Piotton, Françoise; Leuba, Geneviève; Herrmann, François R; Savioz, Armand

    2016-01-01

    The amyloid-β peptide or Aβ is the key player in the amyloid-cascade hypothesis of Alzheimer's disease. Aβ appears to trigger cell death but also production of double-strand breaks (DSBs) in aging and Alzheimer's disease. All-trans retinoic acid (RA), a derivative of vitamin A, was already known for its neuroprotective effects against the amyloid cascade. It diminishes, for instance, the production of Aβ peptides and their oligomerisation. In the present work we investigated the possible implication of RA receptor (RAR) in repair of Aβ-induced DSBs. We demonstrated that RA, as well as RAR agonist Am80, but not AGN 193109 antagonist, repair Aβ-induced DSBs in SH-SY5Y cells and an astrocytic cell line as well as in the murine cortical tissue of young and aged mice. The nonhomologous end joining pathway and the Ataxia Telangiectasia Mutated kinase were shown to be involved in RA-mediated DSBs repair in the SH-SY5Y cells. Our data suggest that RA, besides increasing cell viability in the cortex of young and even of aged mice, might also result in targeted DNA repair of genes important for cell or synaptic maintenance. This phenomenon would remain functional up to a point when Aβ increase and RA decrease probably lead to a pathological state.

  11. Gremlin-mediated decrease in bone morphogenetic protein signaling promotes aristolochic acid-induced epithelial-to-mesenchymal transition (EMT) in HK-2 cells.

    PubMed

    Li, Yi; Wang, Zihua; Wang, Shuai; Zhao, Jinghong; Zhang, Jingbo; Huang, Yunjian

    2012-07-16

    Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that bone morphogenetic protein-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (10 μmol/L) for periods up to 72 h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and α-smooth muscle actin (α-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and protein expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to 10 μmol/L AA increased the mRNA and protein expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 protein levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin

  12. Low simvastatin concentrations reduce oleic acid-induced steatosis in HepG2 cells: An in vitro model of non-alcoholic fatty liver disease

    PubMed Central

    ALKHATATBEH, MOHAMMAD J.; LINCZ, LISA F.; THORNE, RICK F.

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is an inflammatory condition caused by hepatic lipid accumulation that is associated with insulin resistance, diabetes and metabolic syndrome. Although statins should be used with caution in liver diseases, they are increasingly investigated as a possible treatment for NAFLD. The present study recreated an in vitro model of NAFLD using HepG2 cells exposed to oleic acid (OA), which was used to quantify OA-induced lipid accumulation in HepG2 cells treated with various concentrations of simvastatin. In addition, the effect of simvastatin on HepG2 cell morphology and microparticle generation as a marker of cell apoptosis was assessed. OA-induced lipid accumulation was quantified by Oil Red O staining and extraction for optical density determination. Stained lipid droplets were visualized using phase contrast microscopy. Furthermore, HepG2 cell-derived microparticles were counted by flow cytometry subsequent to staining for Annexin V. HepG2 cells treated with 0–1 mM OA showed dose-dependent lipid accumulation. Treatment of HepG2 cells with increasing concentrations of simvastatin followed by treatment with 1 mM OA showed that low simvastatin concentrations (4–10 µM) were able to reduce lipid accumulation by ~40%, whereas high simvastatin concentrations (20 and 30 µM) induced apoptotic changes in cell morphology and increased the production of Annexin V+ microparticles. This suggests that low simvastatin doses may have a role in preventing NAFLD. However, further investigations are required to confirm this action in vivo and to determine the underlying mechanism by which simvastatin reduces hepatic steatosis. PMID:27073470

  13. Spergularia marina Induces Glucagon-Like Peptide-1 Secretion in NCI-H716 Cells Through Bile Acid Receptor Activation

    PubMed Central

    Kim, Kyong; Lee, Yu Mi; Rhyu, Mee-Ra

    2014-01-01

    Abstract Spergularia marina Griseb. (SM) is a halophyte that grows in mud flats. The aerial portions of SM have been eaten as vegetables and traditionally used to prevent chronic diseases in Korea. However, there has been no scientific report that demonstrates the pharmacological effects of SM. Glucagon-like peptide-1 (GLP-1) is important for the maintenance of glucose and energy homeostasis through acting as a signal in peripheral and neural systems. To discover a functional food for regulating glucose and energy homeostasis, we evaluated the effect of an aqueous ethanolic extract (AEE) of SM on GLP-1 release from enteroendocrine NCI-H716 cells. In addition, we explored the Takeda G-protein-coupled receptor 5 (TGR5) agonist activity of AEE-SM in Chinese hamster ovary (CHO)-K1 cells transiently transfected with human TGR5. As a result, treatment of NCI-H716 cells with AEE-SM increased GLP-1 secretion and intracellular Ca2+ and cyclic AMP (cAMP) levels in a dose-dependent manner. Transfection of NCI-H716 cells with TGR5-specific small interference RNA inhibited AEE-SM-induced GLP-1 secretion and the increase in Ca2+ and cAMP levels. Moreover, AEE-SM showed that the TGR5 agonist activity in CHO-K1 cells transiently transfected with TGR5. The results suggest that AEE-SM might be a candidate for a functional food to regulate glucose and energy homeostasis. PMID:25260089

  14. AP-1 Inhibition by SR 11302 Protects Human Hepatoma HepG2 Cells from Bile