BBA - General Subjects (v.1810, #5)

Siphonaxanthin, a marine carotenoid from green algae, effectively induces apoptosis in human leukemia (HL-60) cells by Ponesakki Ganesan; Kenji Noda; Yuki Manabe; Takeshi Ohkubo; Yukihisa Tanaka; Takashi Maoka; Tatsuya Sugawara; Takashi Hirata (497-503).
Bioactive marine molecules have recently received considerable attention for their nutraceutical characteristics. Considering the ever-increasing demand of nutraceuticals for anti-cancer therapy, we investigated the apoptosis-inducing effects of marine carotenoids, including siphonaxanthin, on human leukemia (HL-60) cells.Apoptotic effects were evaluated by cell viability assay, TUNEL assay, and caspase-3 activity. The expression of apoptosis-inducing death receptor-5 (DR5), Bcl-2 and Bax were assayed by Western blot analysis, and mRNA expression of GADD45α was assayed by quantitative RT-PCR analysis.Siphonaxanthin potently inhibited the viability of HL-60 cells compared with the other carotenoids evaluated. In comparison with fucoxanthin, siphonaxanthin at a concentration of 20 μM markedly reduced cell viability (p  < 0.05) as early as within 6 h of treatment. The effective apoptotic activity of siphonaxanthin was observed by increases in TUNEL-positive cells, and by increased chromatin condensation in HL-60 cells. This induction of apoptosis was associated with the decreased expression of Bcl-2, and the subsequently increased activation of caspase-3. In addition, siphonaxanthin up-regulated the expression of GADD45α and DR5.These data suggest that the dietary carotenoid siphonaxanthin could be potentially useful as a chemo-preventive and/or chemotherapeutic agent.Our findings demonstrate for the first time the novel functional property of siphonaxanthin as a potent inducer of apoptosis in HL-60 cells.► The present findings demonstrate for the first time the novel functional property of siphonaxanthin as a potent inducer of apoptosis in HL-60 cells. ► The apoptosis-inducing effect of siphonaxanthin is stronger than fucoxanthin. ► The induction of apoptosis by siphonaxanthin is associated with the up-regulation of tumor selective transmembrane receptor, DR5.
Keywords: Carotenoid; Siphonaxanthin; Fucoxanthin; HL-60; Apoptosis;

p53 in trichostatin A induced C6 glioma cell death by Ya-Fen Hsu; Joen-Rong Sheu; George Hsiao; Chien-Huang Lin; Tsai-Hsing Chang; Pei-Ting Chiu; Chun-Yu Wang; Ming-Jen Hsu (504-513).
Histone deacetylase (HDAC) inhibitors were demonstrated to induce cell cycle arrest, promote cell differentiation or apoptosis, and inhibit metastasis. HDAC inhibitors have thus emerged as a new class of anti-tumor agents for various types of tumors. However, the mechanisms by which HDAC inhibition-induced cell death remain to be fully defined.In the present study, we explored the apoptotic actions of trichostatin A (TSA), a HDAC inhibitor, in C6 glioma cells.TSA activated p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation and activation. P53, a proapoptotic transcription factor, in turn transactivated the expression of a proapoptotic protein, Bax. In addition, survivin, a member of inhibitor of apoptotic protein, was significantly decreased in TSA-treated C6 cells. P53 recruited to the endogenous survivin promoter region was increased and accompanied by decreasing recruitment of SP1 in response to TSA. TSA was also shown to induce IKK dephosphorylation and to suppress NF-κB reporter activity.TSA may cause C6 cell apoptosis through activating p38MAPK–p53 cascade resulting in Bax expression and survivin suppression. Negative regulation of IKK–NF-κB signaling may also lead to p53 activation and contribute to TSA apoptotic actions.TSA-induced p53 activation may occur through p53 modification by phosphorylation or by acetylation via IKK inactivation. The present study delineates, in part, the signaling pathways involved in TSA-induced glioma cell death.Display Omitted► Trichostatin A causes glioma cell apoptosis through activating p38MAPK–p53 cascade ► Trichostatin A increases p53 phosphorylation and acetylation ► Trichostatin A increases Bax, but decreases Bcl-2, Bcl-xl and survivin levels
Keywords: Trichostatin A; Histone deacetylase; p53; Survivin; Apoptosis;

Binding affinities of CRBPI and CRBPII for 9-cis-retinoids by Maureen A. Kane; Frank V. Bright; Joseph L. Napoli (514-518).
Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.We have determined apparent dissociation constants, K′ d , through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.CRBPI and CRBPII, respectively, bind 9-cis-retinol (K′ d , 11 nM and 68 nM) and 9-cis-retinal (K′ d , 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.► K d values for 9-cis-retinoids and CRBPI and CRBPII. ► Accuracy ensured by non-linear regression analysis for all ligands. ► Concentrations of retinoid binding proteins were an order of magnitude lower than used previously, to enhance accuracy.
Keywords: Cellular retinol binding-protein; Retinol; Retinal; Retinoic acid; Vitamin A; Retinoid binding affinities;

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937 by Hidehiko Akiyama; Masahiro Endo; Taei Matsui; Itsurou Katsuda; Nobuhiko Emi; Yasuko Kawamoto; Takaaki Koike; Hidehiko Beppu (519-525).
Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine.Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells.Nuclear damage, DNA fragmentation, was observed by Wright–Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10 μg/mL of agaritine for 48 h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment.These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria.This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.► Effects of agaritine from Agaricus blazei Murrill on U937 cells were studied. ► Agaritine induced DNA fragmentation, annexin V expression and cytochrome c release. ► Caspase-3, -8 and -9 activities gradually increased after agaritine treatment. ► These results suggest that agaritine moderately induces apoptosis in U937 cells.
Keywords: Agaritine; Annexin V; Apoptosis; Caspase; Cytochrome c; U937 cell;

In vitro studies on hyperbaric oxygen (HBO) therapy suggest that HBO may cause DNA damage, but this has not been evaluated using endothelial cells.Human umbilical cord endothelial cells (HUVECs) were exposed either to H2O2 or to HBO for 90 min, with or without subsequent H2O2 exposure. Measurements included the comet assay for DNA damage, and reduced and oxidised glutathione levels.HUVECs showed sensitivity to H2O2 (EC50 of 0.2 mM for DNA migration). A single 90 min HBO treatment at 2.2 ATA caused a statistically significant (ANOVA, P  < 0.05) increase of DNA migration in HUVECs to 6.8 ± 0.3% (mean ± SEM, n  = 8), which returned to normal levels (4.9 ± 0.1%, n  = 6) after 24 h. Further exposure to 0.2 mM H2O2 after HBO treatment significantly increased the DNA migration in HBO-treated cells immediately post-treatment; but 24 h later the cells showed 22% less DNA damage and higher glutathione than controls.A single HBO exposure causes limited DNA damage to HUVECs, which repairs quickly. HBO treatment protects against H2O2-induced DNA damage and involves cellular glutathione.Endothelial cells are unlikely to be compromised during HBO therapy.► Novel use of human umbilical cord endothelial cells, HUVECs, with hyperbaric oxygen. ► HUVECs are a good model for measuring DNA damage and respond to H2O2. ► A single 90 min HBO treatment at 2.2 ATA causes limited DNA damage to HUVECs. ► Cells quickly repaired within 24 h of HBO treatment. ► HBO treatment protects HUVECs from H2O2-induced DNA damage and involves glutathione.
Keywords: DNA damage; Comet assay; Human umbilical vein endothelial cells (HUVECs); Hyperbaric oxygen; Glutathione; Hydrogen peroxide;

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins by Lee Hedden; Cyril H. Benes; Stephen P. Soltoff (532-542).
The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca2+]i. We report that some agents that can block P2X7R receptors also promote diverse P2X7R-independent effects on cell signaling.We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X7R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated 45Ca2+ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X7R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.Several agents used as P2X7R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.Display Omitted► Agents that block P2/P2X7 receptors promote the phosphorylation of cell signaling proteins in a cell-type specific manner. ► ERK, Src, PKC, and CDCP1 are among the proteins that are phosphorylated/activated. ► Phosphorylation events promoted by P2R blockers are independent of the P2X7 receptor.
Keywords: Parotid; ERK1/2; Src; CDCP1; P2 receptor; Phosphorylation;

Abnormal subcellular localization of AQP5 and downregulated AQP5 protein in parotid glands of streptozotocin-induced diabetic rats by Di Wang; Zhenfang Yuan; Noriko Inoue; Gota Cho; Masayuki Shono; Yasuko Ishikawa (543-554).
The mechanisms underlying diabetic xerostomia have not been clarified in relation with aquaporin-5 (AQP5) subcellular localization in salivary glands.Western blotting, real-time PCR, and immunocytochemistry were used to analyse AQP5 protein levels and mRNA expression. AQP5 protein levels were measured in the apical plasma membrane (APM) and detergent-insoluble fraction prepared from streptozotocin-diabetic rat parotid glands.Despite an increase in AQP5 mRNA, AQP5 protein levels were decreased in diabetic parotid glands compared with controls. Immunohistochemical studies indicated that AQP5, under unstimulated conditions, colocalised with flotillin-2 and GM1 with a diffuse pattern in the apical cytoplasm of acinar and duct cells in both control and diabetic rats. Ten minutes after intravenous injection of muscarinic agonist cevimeline, AQP5 was dramatically increased together with flotillin-2 and GM1 in the APM of parotid acinar and duct cells of control but not diabetic rats. Sixty minutes after injection, AQP5 was located in a diffuse pattern in the apical cytoplasm in both rats. Treatment of the parotid tissues with cevimeline for 10 min increased the Triton X-100 solubility of AQP5 in control but not diabetic rats. Administration of insulin to diabetic rats tended to restore the cevimeline-induced translocation of AQP5.Lack of AQP5 translocation in the salivary gland in response to a muscarinic agonist and downregulation of AQP5 protein might lead to diabetic xerostomia.General significanceCevimeline is useful to cure diabetic xerostomia under insulin administration.► A common complaint associated with diabetes mellitus is xerostomia. ► Xerostomia leads to a vicious cycle of morbidity. ► Lack of AQP5 translocation in salivary gland and downregulation of AQP5 lead to diabetic xerostomia. ► Cevimeline is useful to cure diabetic xerostomia under insulin administration.
Keywords: Type 1 diabetes; Aquaporin-5; Lipid raft; Parotid gland; Xerostomia; Translocation;

Time dependent changes in aortic tissue during cold storage in physiological solution by M.G. Ghosn; M. Mashiatulla; M.A. Mohamed; S. Syed; F. Castro-Chavez; J.D. Morrisett; K.V. Larin (555-560).
Stored vascular tissues are employed in biomedical research for studies in imaging, in biomechanics, and/or in assessing vessel diseases. In the present study, the stability of aortic tissue in phosphate buffer saline (PBS) at 4 °C was monitored over a course of 10 days as determined by the rate of glucose permeation measured by optical coherence tomography (OCT) and validated by histology.The initial mean permeability through fresh porcine aorta was (2.32 ± 0.46) × 10− 5  cm/s (n  = 5); after maintaining the tissue at 4 °C for 10 days, the mean rate was (7.37 ± 0.41) × 10− 5  cm/s (n  = 4), an increase of nearly 300%. A z-test verified that a significant change in the permeability rate (p  < 0.05) had occurred after 4 days of 4 °C storage. Histology was used to quantify changes in tissue pore area. The increase in average pore area paralleled the increase in permeability rate over 10 days.These results suggest that (1) the structural integrity of aortic tissue at 4 °C is retained for at least the first three days after resection and (2) OCT is a powerful technology well suited for evaluating tissue structural integrity over time.Functional OCT imaging provides for a noninvasive and quantitative technique in determining the structural integrity of aortic tissue stored at 4 °C. This modality may be used for assessing the efficacy of other preservation techniques.► Optical coherence tomography (OCT) is a noninvasive imaging technique. ► OCT used to study integrity of tissue structure during 10 day storage at 4 °C. ► OCT monitors the permeation of analytes through biological tissue. ► Increasing permeability rate was correlated to an increase in tissue pore size.
Keywords: Permeability rate; Hypothermal preservation; Cold tissue storage; Porcine aorta; Pore size;

AtoSC two-component system is involved in cPHB biosynthesis through fatty acid metabolism in E. coli by Evaggelos C. Theodorou; Marina C. Theodorou; Dimitrios A. Kyriakidis (561-568).
We have shown previously that AtoSC two-component system regulates the biosynthesis of E. coli cPHB [complexed poly-(R)-3-hydroxybutyrate].The AtoSC involvement on fatty acids metabolism, towards cPHB synthesis, was studied using cPHB determination, gene expression, and fatty acid metabolic pathways inhibitors.Deletion of the atoDAEB operon from the E. coli genome resulted in a consistent reduction of cPHB accumulation. When in ΔatoDAEB cells, the atoDAEB operon and the AtoSC system were introduced extrachromosomally, a significant enhancement of cPHB levels was observed. Moreover, the introduction of a plasmid with atoSC genes regulated positively cPHB biosynthesis. A lesser cPHB enhancement was triggered when plasmids carrying either atoS or atoC were introduced. The intracellular distribution of cPHB was regulated by AtoSC or AtoC according to the inducer (acetoacetate or spermidine). Blockage of β-oxidation by acrylic acid reduced cPHB levels, suggesting the involvement of this pathway in cPHB synthesis; however, the overproduction of AtoSC or its constituents separately resulted in cPHB enhancement. Inhibition of fatty acid biosynthesis by cerulenin resulted to a major cPHB reduction, indicating the contribution of this pathway in cPHB production. Inhibition of both β-oxidation and fatty acid biosynthesis reduced dramatically cPHB, suggesting the contribution of both pathways in cPHB biosynthesis.Short fatty acid catabolism (atoDAEB operon) and fatty acids metabolic pathways participate in cPHB synthesis through the involvement of AtoSC system.The involvement of the AtoSC system in the fatty acids metabolic pathways interplay towards cPHB biosynthesis provides additional perceptions of AtoSC role on E. coli regulatory biochemical processes.► cPHB biosynthesis by β-oxidation, fatty acid synthesis, and atoDAEB operon. ► AtoSC involvement in fatty acid metabolism interplay towards cPHB regulation. ► Identification of new downstream targets of the AtoSC two-component system. ► AtoSC constituents separately up-regulate cPHB through fatty acid metabolism.
Keywords: AtoS–AtoC; atoDAEB; Two-component system; cPHB; β-oxidation; Fatty acid biosynthesis;

Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP by Wen-Qiang Chang; Xiu-Zhen Wu; Ai-Xia Cheng; Li Zhang; Mei Ji; Hong-Xiang Lou (569-576).
Retigeric acid B (RAB), a triterpene acid isolated from Lobaria kurokawae exerts antifungal effect. The present study was designed to elucidate the underlying mechanisms by which RAB regulates the proliferation and cell death of Candida albicans.We measured the metabolic activity of C. albicans with WST1 Cell Proliferation and Cytotoxicity Assay Kit, analyzed the cell cycle by flow cytometry, visualized the ultrastructure by transmission electron microscopy (TEM) and investigated the apoptosis and necrosis induced by RAB using confocal microscopy. The reactive oxygen species (ROS) accumulation was determined by spectrophotometry, flow cytometry and fluorescent microscopy. The mtΔψ was detected using flow cytometry. And the levels of intracellular cAMP and ATP were measured with cAMP ELISA and ATP Assay Kits, respectively.The proliferation of the yeasts was blocked in G2/M phase by a low dose of RAB treatment and in G1 phase at high concentration. When cultured in phosphate buffered saline (PBS) deprived of energy source, yeasts displayed the phenotype of death caused by accumulated ROS, mtΔψ hyperpolarization and dramatic decrease in ATP level in the presence of high dose of RAB.RAB inhibits the growth of C. albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB. Our findings provide a novel molecular mechanism for exploring possible applications of lichen derived metabolites in fighting fungal infection in humans.►Retigeric acid B (RAB), a triterpene acid isolated from the lichen species Lobaria kurokawae inhibits the growth of Candida albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB.
Keywords: Candida albicans; Retigeric acid B; Cell cycle; Reactive oxygen species; cAMP;