Antitumor effects of human recombinant interleukin-1α and etoposide against human tumor cells: mechanism for synergismin vitro and activityin vivo

Recombinant human interleukin 1α (rh IL-1α) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell linesin vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1α. The purposes of this study were to test our hypothess that these events were critical to the synergy between rhIL-1α and VP-16, to determine whether rhIL-1α and VP-16 synergize to increase superoxide (SO) anion radical productionin vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combinaton against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1ra) before exposure to rhIL-1α, VP-16 and rhIL-1α plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1α, VP-16, and rhIL-1α+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1α and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1α and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1α and VP-16. However, when A375/S0D15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfecton were exposed to rhIL-1α and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1α and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1α and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combnation of IL-1α and VP-16 might prove useful for the treatment of malignant diseasein vivo, if the increased toxicity can be reduced or managed. The US Government’s right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged.     

Effects of 5-bromo-2′-deoxyuridine on arachidonic acid metabolism of neuroblastoma and leukemia cells in culture: A possible role of endogenous prostaglandins in tumor cell proliferation and differentiation

Effects of 5-bromo-2′-deoxyuridine (BrdU) were studied on two neuroblastoma and two leukemia cell lines, in terms of the relationship between prostaglandin (PG) synthesis and cell growth/differentiation. After treatment with BrdU (5 μg/ml), cell growth of the 4 cell lines was inhibited and one neuroblastoma cell line (GOTO) showed flattened morphology with positive S-100 protein, one of the differentiation markers for Schwann or glial cells. In the 4 cell lines, BrdU treatment reduced [1-¹⁴C]-arachidonic acid incorporation into phosphatidylinositol and phosphatidylethanolamine and was associated with an increase into phosphatidylcholine and triglyceride. BrdU treatment also increased fractions of 6-keto-PGF_1α and PGF_2α , with a decreased TXB₂ fraction. The decreased ratio of TXB₂ /6-keto-PGF_1α or increased 6-keto-PGF_1α fraction correlated significantly with cell growth inhibition, suggesting that the changes in the balance of endogenous PGs might be associated with BrdU-induced cell growth inhibition with or without differentiation of neuroblastoma and leukemia cells in culture.     

Effects of recombinant human interleukin 1a and interleukin 1β on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Recombinant human interleukin 1 (rhIL-1)α and rhIL-1β were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1α and rhIL-1β was compared in terms of the units which induced half-maximal [³H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1α and rhIL-1β significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-lα and rhIL-1β markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1α and rhIL-1β increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1α and rhIL-1β have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.     

Effect of recombinant human interleukin 1β (rhIL-1β) on amino acid flux in the isolated perfused rat liver

Summary We studied the effect of recombinant human IL-1 (rhIL-1) on hepatic amino acid (AA) flux in the isolated perfused rat liver model. Two experimental groups were used — a control group (n = 5) and a rhIL-1-treated group (n = 5). IL-1 was added to the perfusate in two successive boluses of 0.1µg and 0.9µg, respectively 35 min (final concentration 0.67 ng/ml) and 60 min (6 ng/ml) after beginning the perfusion. In the IL-1 treated group, a reduction in flux was observed for only three AA, alanine, phenylalanine and serine. Glucose and urea production in the IL-1-treated group was slightly but not-significantly lower than in the controls.rhIL-1 thus has only minor direct effects on AA flux and gluconeogenesis in the liver and cannot therefore be held responsible for the increase in hepatic amino acid uptake during stress.     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Antitumor effect of combination of murine recombinant interferon β, murine recombinant interferon γ and human recombinant interleukin-2 in MethA-bearing mice

Summary We have previously reported that the combination of murine recombinant interferon (Mu-rIFN) with murine recombinant interferon (Mu-rIFN) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN with Mu-rIFN on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 × 10³ U or 5 × 10⁴ U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 × 10⁵ U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 × 10³ U and 5 × 10⁵ U) in addition to the combination of IFN and IFN showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN and IFN had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN and was effective for cancer treatment.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Analysis of isoform specific ERK signaling on the effects of interleukin-1β on COX-2 expression and PGE2 production in human chondrocytes

The MAPK/ERK pathway is involved in IL-1β-induced cyclooxygenase (COX-2) expression and prostaglandin E2 (PGE2) production; two factors that play important roles in OA pathogenesis. In the present study, we find that IL-1β induced COX-2 expression and PGE2 production in human chondrocytes via a process that required the activation of the MAPK/ERK pathway. To evaluate the respective roles and relationship of ERK1 and ERK2 on IL-1β induced COX-2 expression and PGE2 production, small interfering RNA was used to knockdown ERK1, ERK2 or both in human chondrocytes. COX-2 expression and PGE2 production were significantly suppressed to a similar degree by the silencing of ERK1 or ERK2 alone. Moreover, the combined knockdown displayed a synergistic effect. Simultaneously, Western blotting indicated that the knockdown of ERK1 or ERK2 down regulated phospho-ERK1 and ERK1 or phospho-ERK2 and ERK2 levels, respectively. No significant compensatory mechanism through the upregulation of the other phospho-ERK and ERK isoform was observed. The combined silencing suppressed both phospho-ERK1/2 and ERK1/2. In conclusion, each ERK isoform similarly influenced IL-1β-mediated COX-2 expression and PGE2 production in human chondrocytes, and ERK1 and ERK2 displayed synergistic effects. Although, inhibition of both ERK1 and ERK2 would be a more effective, each ERK isoform may sufficiently regulate these effects in human chondrocytes. ERK1 or ERK2 may be potential therapeutic target for the inflammatory process of OA.     

IL-1β and TNF-α alter the glycophenotype of primary human chondrocytes in vitro

Despite the significance of glycoproteins for extracellular matrix assembly in cartilage tissue, little is known about the regulation of the chondrocyte glycophenotype under inflammatory conditions. The present study aimed to assess the effect of IL-1β and TNF-α on specific features of the glycophenotype of primary human chondrocytes in vitro. Using LC-MS, we found that both cytokines increased overall sialylation of N- and O-glycans and induced a shift towards α-(2→3)-linked sialic acid residues in chondrocyte glycoproteins. These results were supported by quantitative PCR showing increased expression of α-(2→3) sialyltransferases in treated cells. Moreover, we found that both IL-1β and TNF-α induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans. In contrast, core α-(1→6)-fucosylation of chondrocyte N-glycans was found to be reduced particularly by TNF-α. In summary, inflammatory conditions induce specific alterations of the chondrocyte glycophenotype which might affect cell-matrix interactions or the function of endogenous lectins.     

Effect of surfactant pluronic F-68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN-γ in mild operating conditions

The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation. Under these conditions, a dose-dependent effect of PF-68 (0-0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF-68 led to increased IFN-γ production as a result of both higher cell densities and a higher specific production rate of IFN-γ. If cells were grown with agitation, lack of PF-68 in the culture medium decreased the fraction of the fully glycosylated IFN-γ glycoform (2N) from 80% to 65-70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF-68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.     

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE₂, PGD₂, and PGF_2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A₂ (PLA₂) isozymes revealed that cytosolic PLA₂α, but not calcium-independent PLA₂s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.     

Interleukins 1α and 1β as regulators of steroidogenesis in human NCI-H295R adrenocortical cells

Inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) regulate the activity of the hypothalamo-pituitary-adrenal (HPA) axis at several levels. Although hypothalamic CRH secretion may be the primary mechanism by which these cytokines activate the HPA axis, IL-1 expression is increased within the adrenal glands in models for systemic inflammation, and IL-1 may augment adrenal glucocorticoid production. Our aim was to investigate the direct effects of IL-1α and IL-1β on adrenal steroidogenesis and expression of three key steroidogenic genes in human adrenocortical cells using the NCI-H295R cell line as a model. mRNAs encoding receptors for IL-1, TNF-α, and leukemia inhibitory factor (LIF) were detectable in the cell line (Affymetrix microarray analysis). Both IL-1α and IL-1β increased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate production, and the accumulation of mRNAs for steroidogenic acute regulatory protein (STAR), 17α-hydroxylase/17,20-lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase 2 (HSD3B2) in these cells (P<0.05 for all). Both ILs augmented TNF-α- and LIF-induced STAR and CYP17A1 mRNA accumulation, and TNF-α-induced cortisol production (P<0.05 for all). Both ILs also increased the apoptotic index of the cells (P<0.05), which was efficiently neutralized by their specific antibodies. The IL-induced changes in the STAR, HSD3B2, and CYP17A1 protein levels were not as evident as those in the respective mRNA levels. In conclusion, the combined effect of inflammatory cytokines at the adrenal level in acute or chronic inflammatory states could significantly stimulate glucocorticoid production, and thus explain the observed discrepancy between the cortisol and ACTH concentrations sometimes seen in sepsis and chronic inflammatory states.     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

Retinoic acid receptor isoform RARγ 1: an antagonist of the transactivation of the RARβ RARE in epithelial cell lines and normal human keratinocytes

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR₁ and RAR₂ isoforms, but not by RAR₁. On the contrary, this latter isoform behaved towards the RAR RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR₁ and RAR₂. RAR₁ also behaved as an antagonist of the transactivation produced by cotransfected RXR. The natural RAR gene promoter or RAR RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR₁. It was, however, possible to activate to a certain extent RAR RARE-reporter constructs in NHK by co-transfecting RAR₁, RAR₂ or RXR. The antagonist behavior of RAR₁ towards the RAR RARE may explain why in certain cell types such as keratinocytes, RAR is neither expressed nor induced by RA.Abbreviations DMEM Dulbecco's modified Eagle medium - DMSO dimethyl sulfoxide - FCS fetal calf serum - MEM minimal Eagle medium - NHK normal human keratinocyte - RA retinoic acid - RAR retinoic acid receptor - RARE retinoic acid responsive element - TRE thyroid responsive element - VDRE vitamin D response element - RXR retinoid X receptor     

Modulatory effect of interleukin-1α on expression of structural matrix proteins,MMPs and TIMPs in human cardiac myofibroblasts: Role of p38 MAP kinase

The proinflammatory cytokine interleukin-1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac myofibroblasts (CMF), or the role of p38 mitogen-activated protein kinase (MAPK). We used a focused RT-PCR microarray to investigate the effects of IL-1α on expression of 41 ECM genes in CMF cultured from different patients, and explored regulation by p38 MAPK.IL-1α (10 ng/ml, 6 h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin. However, it induced marked increases in expression of specific ECM proteases, including matrix metalloproteinases MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B) and MMP-10 (stromelysin-2). Conversely, IL-1α reduced mRNA and protein expression of ADAMTS1, a metalloproteinase that suppresses neovascularization. IL-1α increased expression of TIMP-1 slightly, but not TIMP-2. Data for MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and ADAMTS1 were confirmed by quantitative real-time RT-PCR. Tumor necrosis factor-alpha (TNFα), another important myocardial proinflammatory cytokine, did not alter expression of these metalloproteinases. IL-1α strongly activated the p38 MAPK pathway in human CMF. Pharmacological inhibitors of p38-α/β (SB203580) or p38-α/β/γ/δ (BIRB-0796) reduced MMP-3 and ADAMTS1 mRNA expression, but neither inhibitor affected MMP-9 levels. MMP-1 and MMP-10 expression were inhibited by BIRB-0796 but not SB203580, suggesting roles for p38-γ/δ.In summary, IL-1α induces a distinct pattern of ECM protein and protease expression in human CMF, in part regulated by distinct p38 MAPK subtypes, affirming the key role of IL-1α and CMF in post-infarction cardiac remodeling.     

Induction of aromaticl-amino acid decarboxylase mRNA by interleukin-1β and prostaglandin E2 in PC12 cells

Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1- and prostaglandin (PG) E₂ in PC12 cells. The cells were treated with different doses of IL 1- and PGE₂ for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1- and PGE₂ produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE₂ formation, its effects may be occurring by increasing level of PGE₂.Abbreviations AADC aromatic 1-amino acid decarboxylase - IL-1 interleukin 1 - PGE₂ prostaglandin E₂ - GITC guanidinium isothiocyanate - DEPC diethyl pyrocarbonate - MOPS 3-(4-morpholino)propanesulfonic acid - SSPE 0.18M NaCl, 0.001M sodium phosphate, and 0.001M EDTA Special issue dedicated to Dr. Bernard W. Agranoff.     

Role of SIRT1 in Streptococcus pneumoniae-induced human β-defensin-2 and interleukin-8 expression in A549 cell

Streptococcus pneumoniae is an important pathogen of pneumonia in human. Human alveolar epithelium acts as an effective barrier and is an active participant in host defense against invasion of bacterial by production of various mediators. Sirtuin 1 (SIRT1), the prototypic class III histone deacetylase, is involved in the molecular control of lifespans and immune responses. This study aimed at examining the role of SIRT1 in mediating S. pneumoniae-induced human β-defensin-2 (hBD2) and interleukin-8(IL-8) expression in the alveolar epithelial cell line A549 and the underlying mechanisms involved. A549 cells were infected with S. pneumoniae for indicated times. Exposure of A549 cells to S. pneumoniae increased the expressions of SIRT1 protein, hBD2 and IL-8 mRNA, and protein. The SIRT1 activator resveratrol enhanced S. pneumoniae-induced gene expression of hBD2 but decreased IL-8 mRNA levels. Blockade of SIRT1 activity by the SIRT1 inhibitors nicotinamide reduced S. pneumoniae-induced hBD2 mRNA expression but increased its stimulatory effects on IL-8 mRNA. S. pneumoniae-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SIRT1 expression was attenuated by selective inhibitors of ERK and p38 MAPK. The hBD2 mRNA production was decreased by pretreatment with p38 MAPK inhibitor but not with ERK inhibitor, whereas the IL-8 mRNA expression was controlled by phosphorylation of ERK. These results suggest that SIRT1 mediates the induction of hBD2 and IL-8 gene expression levels in A549 cell by S. pneumoniae. SIRT1 may play a key role in host immune and defense response in A549.     

Ascorbate and glutathione metabolism in two sunflower cell lines of differing α-tocopherol biosynthetic capability

The natural antioxidants, tocopherols and ascorbate (ASC), are of great interest in terms of human health, because of their role in the prevention of chronic diseases. In cell metabolism, tocopherols are the major lipid-soluble antioxidants, whereas ASC and glutathione (GSH) are hydro-soluble antioxidants. These three metabolites cooperate in scavenging for oxygen radicals and protecting cell membranes. ASC and GSH are required in the process of regeneration of tocopherol from its α-cromanoxyl radical, while, GSH donates electrons for the reduction of dehydroascorbate (DHA), the fully oxidised form of ASC. Two cell lines of sunflower (Helianthus annuus L. cv Gloriasol) with differing capability to synthesise α-tocopherol were identified. In spite of the differing content of α-tocopherol (almost threefold higher in the high synthesising cell line, HT, than in the low synthesising one, LT), the cell lines have comparable growth curves. In the cells collected in the stationary phase, the ASC and GSH pools are also significantly higher in the HT cells than in the LT cells. On the other hand, the enzymes responsible for H₂O₂ scavenging and ASC and GSH recycling had higher activity in the LT than in the HT cells. The cooperation between the three antioxidant systems in the maintenance of the cellular redox balance is discussed, as well as the possible utilisation of the HT cell line for the in vitro production of natural antioxidants.