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Biochemical Pharmacology (v.75, #3)

Editorial Advisory Board (pp. iii).

The interplay between viruses and innate immune signaling: Recent insights and therapeutic opportunities by Leonie Unterholzner; Andrew G. Bowie (pp. 589-602).

The immediate response to viral infection relies on pattern-recognition receptors (PRRs), most prominently the Toll-like receptors (TLRs) and the RNA helicases RIG-I and MDA5, as well as double stranded RNA-dependent protein kinase (PKR) and the DNA receptor, DAI. These PRRs recognize pathogen-associated molecular patterns (PAMPs) such as viral proteins and nucleic acids. The engagement of these receptors then initiates intracellular signaling cascades which ultimately cause the activation of transcription factors and the expression of type I interferons and pro-inflammatory cytokines. This innate response establishes an anti-viral state in the infected cell and its neighbours and alerts immune cells to the danger. In order to establish a productive infection, viruses need to overcome this initial anti-viral response. Evasion of innate immune defences is achieved by means of viral proteins that inhibit the signaling cascades emanating from the PRRs. The same innate signal transduction pathways have been implicated in conditions of sterile inflammation, such as rheumatoid arthritis and multiple sclerosis, and in autoimmunity. Because viral proteins target crucial host proteins involved in these pathways, they can point the way to key drug targets. Further, the viral proteins themselves or derivatives of them may be of use therapeutically to curtail inflammation and autoimmunity.

Keywords: Abbreviations; AdV; adenovirus; ASFV; African swine fever virus; CARD; caspase recruitment domain; CMV; cytomegalovirus; DC; dentritic cell; ds; double-stranded; EBV; Epstein-Barr virus; eIF; eukaryotic initiation factor; EMCV; encephalomyocarditis virus; FADD; Fas-associated via death domain; HCV; hepatitis C virus; HHV; human herpes virus; HIV; human immunodeficiency virus; HSV; herpes simplex virus; IAV; influenza A virus; IFN; interferon; I-κB; inhibitor of nuclear factor κB; IKK; I-κb kinase; IL; interleukin; IPS-1; interferon-β promoter stimulator-1; IRAK; IL-1 receptor associated kinase; IRF; interferon regulatory factor; JEV; Japanese encephalitis virus; JNK; Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MCMV; mouse cytomegalovirus; MDA5; melanoma differentiation-associated gene 5; mDC; myeloid DC; MMTV; mouse mammary tumor virus; MV; measles virus; MyD88; myeloid differentiation primary response gene 88; NAP; NAK-associated protein; NEMO; NF-κB essential modulator; NDV; Newcastle disease virus; NF-κB; nuclear factor κB; PAMP; pathogen-associated molecular pattern; pDC; plasmacytoid DC; PKR; dsRNA-dependent protein kinase; PRR; pattern-recognition receptor; RHIM; RIP homotypic interaction motif; RIG-I; retinoic acid-inducible gene I; RIP; receptor interacting protein; ss; single-stranded; RSV; Rous sarcoma virus; SeV; Sendai virus; TAB; TAK1-binding protein; TAK; TGF-β activated protein kinase; TANK; TRAF family member-associated NF-κB activator; TBK; TANK-binding kinase; TIR; Toll/interleukin-1 receptor; TLR; Toll-like receptor; TNF; tumor necrosis factor; TRAF; tumor necrosis factor receptor-associated factor; TRIF; TIR-domain containing adaptor inducing IFN-β; VACV; vaccinia virus; VSV; vesicular stomatitis virusViruses; Innate immunity; Signaling; Pattern-recognition receptors; Viral evasion

Marine natural products as targeted modulators of the transcription factor NF-κB by Florence Folmer; Marcel Jaspars; Mario Dicato; Marc Diederich (pp. 603-617).

The inducible transcription factor nuclear factor-kappaB (NF-κB) plays an important role in the regulation of immune, inflammatory and carcinogenic responses. While normal NF-κB activation is necessary for cell survival and immunity, deregulated NF-κB expression is a characteristic phenomenon in cancer development, as well as in several inflammatory diseases. Hence, NF-κB has become a major target in drug discovery, and several natural and synthetic compounds have been investigated for their potential to inhibit NF-κB. Here, we discuss the applications of marine natural products, in particular, as novel, potent NF-κB inhibitors. With the oceans covering two-thirds of the Earth's surface, and with the uniqueness of the environmental properties of marine habitats, it is easily understandable that organisms thriving in the oceans constitute a rich source of chemically unique and biomedically powerful secondary metabolites. Since the early 1960s, significant effort has been placed on the pharmacological evaluation of marine secondary metabolites. Noteworthy achievements of this field of biomedically guided marine exploration, a scientific endeavour often referred to as the search for “ Drugs from the Sea”, include the discovery of numerous potent anti-cancer, anti-inflammatory, antimicrobial, and analgesic compounds. The chemical characteristics and molecular targets of marine NF-κB inhibitors discovered to date are presented and discussed in the context of marine chemical ecology.

Keywords: Abbreviations; IκB; inhibitor of NF-κB; IKK; kinase of IκB; MIC; minimal inhibitory concentration; MNP; marine natural product; NF-κB; nuclear factor-κB; PLA; ₂; phospholipase A; ₂; ROS; reactive oxygen species; TNF-α; tumour necrosis factor α; TNFR1; TNF-α receptor 1; TRADD; TNFR1-associated death domain; TRAF2; TNF-receptor-associated factor 2; VEGF; vascular endothelial growth factorNF-kappaB; Natural product; Marine compounds; Cell signalling; Drug discovery

Differential inactivation of polymorphic variants of human O⁶-alkylguanine-DNA alkyltransferase by Qingming Fang; Natalia A. Loktionova; Robert C. Moschel; Sahar Javanmard; Gary T. Pauly; Anthony E. Pegg (pp. 618-626).

The human DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (hAGT) is an important source of resistance to some therapeutic alkylating agents and attempts to circumvent this resistance by the use of hAGT inhibitors have reached clinical trials. Several human polymorphisms in the MGMT gene that encodes hAGT have been described including L84F and the linked double alteration I143V/K178R. We have investigated the inactivation of these variants and the much rarer variant W65C by O⁶-benzylguanine, which is currently in clinical trials, and a number of other second generation hAGT inhibitors that contain folate derivatives ( O⁴-benzylfolic acid, the 3′ and 5′ folate esters of O⁶-benzyl-2′-deoxyguanosine and the folic acid γ ester of O⁶-( p-hydroxymethyl)benzylguanine). The I143V/K178R variant was resistant to all of these compounds. The resistance was due solely to the I143V change. These results suggest that the frequency of the I143V/K178R variant among patients in the clinical trials with hAGT inhibitors and the correlation with response should be considered.

Keywords: Abbreviations; AGT; O; ⁶; -alkylguanine-DNA alkyltransferase; hAGT; human AGT; BG; O; ⁶; -benzylguanine; PaTrin-2; O; ⁶; -(4-bromothenyl)guanine; BF; O; ⁴; -benzylfolic acid; 3FBDG; O; ⁶; -benzyl-3′-; O; -(γ-folyl)-2′-deoxyguanosine; 5FBDG; O; ⁶; -benzyl-5′-; O; -(γ-folyl)-2′-deoxyguanosine; FHMBG; O; ⁶; -[4-[(γ-folyl)-oxymethyl]benzyl]guanine; IPTG; isopropyl β-; d; -thiogalactopyranoside; Tev; tobacco etch virusAlkyltransferase; O; ⁶; -Benzylgunaine; Polymorphisms; Cancer chemotherapy; O; ⁴; -Benzylfolate; DNA repair

Antitumor effect of the angiogenesis inhibitor bevacizumab is dependent on susceptibility of tumors to hypoxia-induced apoptosis by Muthu Selvakumaran; Kang Shen Yao; Michael D. Feldman; Peter J. O’Dwyer (pp. 627-638).

Angiogenesis inhibition has been shown to enhance the therapeutic efficacy of cytotoxic chemotherapy in colorectal cancer. The basis of the contribution of this modality has not been defined fully. To determine the potential role of hypoxia-induced apoptosis, we studied a series of colon cancer cell lines with varying susceptibility to hypoxia. We exposed HT29 and HCT116 colon adenocarcinoma cell lines to sublethal periods of hypoxia three times weekly for 40 exposures, and derived cell lines both more resistant (from HT29) and more sensitive (from HCT116) to hypoxia-induced apoptosis. Both hypoxia-derived cell lines demonstrated more rapid growth than the parental lines when implanted subcutaneously in immunodeficient mice. Treatment of tumor-bearing mice with bevacizumab resulted in depletion of tumor microvasculature, upregulation of Hypoxia-inducible factor-1 alpha (HIF-1α), and increased pimonidazole staining, consistent with an anti-angiogenic effect and induction of hypoxia in tumors derived from all cell lines. The proportion of apoptotic cells was increased in all the treated tumors, and was most pronounced in the bevacizumab-treated HCT116-derived cells. The bevacizumab-treated tumors showed growth delay in HT29 and its derivative, and the parental HCT116. In the hypoxia-sensitive HCT116-derived tumors, marked tumor shrinkage and prolonged growth control occurred. Therefore, bevacizumab treatment is an effective inducer of a hypoxic environment, but the resulting cell kill and tumor shrinkage is determined by the susceptibility of the tumor to apoptosis. The induction of apoptosis by hypoxia may contribute to the benefits of such treatment in the clinical setting.

Keywords: Hypoxia; VEGF; Bevacizumab; Hypoxia-conditioned cell line

Compensatory effects of the human nucleoside transporters on the response to nucleoside-derived drugs in breast cancer MCF7 cells by Pedro Cano-Soldado; Míriam Molina-Arcas; Berta Algueró; Ignacio Larráyoz; M. Pilar Lostao; Anna Grandas; F.Javier Casado; Marçal Pastor-Anglada (pp. 639-648).

Nucleoside transporters (NTs) are involved in the cytotoxicity and transcriptomic response induced by nucleoside analogues. A relationship between the expression of nucleoside transporters and response to therapy has been demonstrated in solid tumours, although the pattern of such expression is highly variable. Thus, a question is whether the transporter expression pattern rather than specific NT proteins might better explain the ability of tumour cells to respond to nucleoside-derived drug therapy. In this study we used the breast cancer cell lines MCF7 and MCF7–hCNT1 (stably transfected with hCNT1) to determine whether hCNT1 expression can complement hENT1 functional loss in the cytotoxicity and transcriptomic response triggered by nucleoside analogues. Expression of hCNT1 slightly increased cell sensitivity to 5′-deoxy-5-fluorouridine (5′-DFUR). Inhibition of the endogenous equilibrative activity blocked 5′-DFUR cytotoxicity in MCF7 cells, but not in MCF7–hCNT1 cells. Moreover, under equilibrative transport inhibition conditions, induction of some transcriptional targets of 5′-DFUR was blocked in MCF7 cells, whereas ENT-inhibition had no effect on the transcriptional response to 5′-DFUR in MCF7–hCNT1 cells. To confirm the role of hCNT1 in 5′-DFUR treatment, a panel of nucleoside derivatives suitable for hCNT1-inhibition was obtained. The molecule T-Ala inhibited hCNT1-mediated transport. Furthermore, the cytotoxic action of 5′-DFUR and the transcriptional changes produced by this nucleoside analogue were partially inhibited by T-Ala in MCF7–hCNT1 cells. These results show a link between NT function and the pharmacogenomic response to nucleoside analogues and further support the hypothesis that the expression pattern rather than specific transporters determines the cytotoxic effect of nucleoside derivatives.

Keywords: hCNT1; 5′-DFUR; Nucleoside transport; Breast cancer; Inhibitor; Nucleoside analogues

Comparative effects of torasemide and furosemide in rats with heart failure by Punniyakoti T. Veeraveedu; Kenichi Watanabe; Meilei Ma; Rajarajan A. Thandavarayan; Suresh S. Palaniyandi; Ken’ichi Yamaguchi; Kenji Suzuki; Makoto Kodama; Yoshifusa Aizawa (pp. 649-659).

It has been reported that torasemide but not furosemide, may block the renin–angiotensin–aldosterone system and therefore it might attenuate myocardial remodeling accompanied by left ventricular (LV) dysfunction. We therefore compared the therapeutic effects of torasemide, a long-acting loop diuretic, and furosemide, a short-acting one, on the progression of LV remodeling in a rat model of chronic heart failure (CHF) after experimental autoimmune myocarditis (EAM). CHF was elicited in Lewis rats by immunization with porcine cardiac myosin. Twenty-eight days after immunization, rats were treated for 28 days with torasemide, furosemide, or vehicle. We investigated the effects on metabolic and neurohumoral parameters, cardiac fibrosis and remodeling in EAM rats. Diuresis was increased dose dependently by both torasemide and furosemide, showed an equipotent natriuretic effect. The urinary potassium excretion was significantly increased with furosemide in comparison to torasemide. Myocardial functional parameters were significantly improved by torasemide. Conversely, these parameters did not change in rats receiving furosemide. Torasemide suppressed LV fibrosis, myocardial protein levels of transforming growth factor-beta1, collagen III, and aldosterone synthase and improved survival rate to the control level, but furosemide did not. Moreover, both pharmacological interventions significantly elevated plasma angiotensin II and decreased atrial natriuretic peptide in a dose-dependent manner. Our results demonstrate that compared with furosemide, torasemide treatment significantly improved survival rate, LV function and ameliorated the progression of cardiac remodeling in rats with CHF after EAM.

Keywords: Experimental autoimmune myocarditis; Remodeling; Fibrosis; Aldosterone; Torasemide; Furosemide

Adenovirus-mediated transfer of siRNA against peroxiredoxin I enhances the radiosensitivity of human intestinal cancer by Bo Zhang; Yan Wang; Kaiyuan Liu; Xaoya Yang; Min Song; Yanyan Wang; Yun Bai (pp. 660-667).

Peroxiredoxin I (Prx-I), a key member of the peroxiredoxin family, reduces peroxides and equivalents through the thioredoxin system. Our previous work has shown that expression of Prx-I in mammalian cells increases following ionizing radiation (IR), and suppression of its expression enhances radiation-induced cell death in vitro, suggesting that inhibition of Prx-I might be an important pretreatment for cancer radiotherapy. To test this hypothesis in vivo, we suppressed the expression of Prx-I in the human intestinal cancer cell line SW480 by adenovirus-mediated transfer of siRNA. Our results showed that expression of Prx-I in SW480 cells was dramatically reduced by recombinant Ad-SiPrxI, which resulted in decreased cell growth and increased cell death by IR. Significantly more cell apoptosis was detected by flow cytometry analysis when Prx-I expression was knocked down. To evaluate the effect of recombinant Ad-SiPrxI in vivo, xenografts were pretreated with adenovirus before IR. Tumor growth in mice was inhibited when the xenografts were pretreated with Ad-SiPrxI before IR. Our results suggest that pretreatment with recombinant adenovirus to inhibit Prx-I expression can enhance the radiosensitivity of cancer cells, and thus might be a potential application in clinical therapy.

Keywords: Peroxiredoxin I; Adenovirus; Ionizing radiation; RNA interference

Cyclooxygenase-2-dependent and -independent inhibition of proliferation of colon cancer cells by 5-aminosalicylic acid by Carmine Stolfi; Daniele Fina; Roberta Caruso; Flavio Caprioli; Massimiliano Sarra; Massimo Claudio Fantini; Angelamaria Rizzo; Francesco Pallone; Giovanni Monteleone (pp. 668-676).

The cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway may have a pathogenic role in colorectal cancer (CRC). Recent studies suggest that 5-aminosalycilic acid (5-ASA) reduces the risk of inflammatory bowel disease-related CRC, but the mechanism by which 5-ASA interferes with CRC cell growth remains unknown. In this study, we have examined whether the negative effect of 5-ASA on CRC cells is dependent on COX-2/PGE2 axis inhibition. We show that 5-ASA down-regulates both constitutive and TNF-α or IL-1β-induced COX-2 in HT-115 and HT-29 cells. Inhibition of COX-2 by 5-ASA occurs at the RNA and protein level, and is associated with a significant decrease in PGE2 synthesis, arrest of growth and enhanced death of CRC cells. However, exogenous PGE2 does not revert the 5-ASA-mediated CRC cell proliferation block. 5-ASA also inhibits the growth of DLD-1, a COX-deficient CRC cell line, thus suggesting that the anti-proliferative effect of 5-ASA on CRC cells is not strictly dependent on the inhibition of COX-2/PGE2. Taken together our data indicate that 5-ASA causes both a COX-2-dependent and -independent inhibition of CRC cell growth.

Keywords: 5-ASA; Colon cancer; COX-2

Regulation of inflammation signalling by resveratrol in human chondrocytes in vitro by Constanze Csaki; Nerses Keshishzadeh; Karoline Fischer; Mehdi Shakibaei (pp. 677-687).

The inflammatory process plays a pivotal role during the pathogenesis of osteoarthritis, dominated by catabolic processes initiated by pro-inflammatory cytokines such as IL-1β. Resveratrol, a natural phytoalexin occurring in various fruits has previously been shown to exhibit anti-inflammatory properties in several cell types. We investigated, whether resveratrol may be a useful blocker of pro-inflammatory cytokine signalling pathways in arthritis.We first examined the effects of resveratrol on the proliferation and production of IL-1β in primary human articular chondrocytes treated with IL-1β in vitro. Resveratrol reversed significantly IL-1β-reduced cell proliferation and blocked IL-1β-stimulated cell membrane bound- and mature IL-1β synthesis in chondrocytes. Furthermore, resveratrol was able to inhibit the IL-1β-induced degradation of mitochondria and apoptosis in chondrocytes in a time-dependent manner. Because caspase inhibitor Z-DEVD-FMK abolished the IL-1β-induced apoptosis in chondrocytes, we examined the effect of resveratrol on the caspase pathway and found that resveratrol blocked the cysteine protease caspase-3 and subsequent cleavage of the DNA repair enzyme PARP. Additionally, resveratrol reversed the IL-1β-induced up-regulation of reactive oxygen species (ROS) in chondrocytes. Finally, we show that resveratrol induced ubiquitin-independent degradation of tumor suppressor gene protein p53 and inhibited p53-induced apoptosis in chondrocytes in a dose-dependent manner.Our results indicate that resveratrol seems to be an effective in vitro anti-inflammatory agent and has a chondroprotective capacity through suppression of (1) IL-1β- (2) ROS- and (3) tumor suppressor protein p53-production. Further studies should be undertaken to define a possible implication of resveratrol in osteoarthritis therapy and cartilage tissue engineering.

Keywords: Chondrocytes; Resveratrol; IL-1β; Apoptosis; Caspase3; p53

(2 R,3 R)-2-(3′,4′-dihydroxybenzyl)-3-(3″,4″-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils by Yi-Jia Huang; Ih-Sheng Chen; Ching-Ping Tseng; Yuan-Ji Day; Yin-Chou Lin; Chang-Hui Liao (pp. 688-697).

This study investigated the mechanism underlying the inhibiting effect of (2 R,3 R)-2-(3′,4′-dihydroxybenzyl)-3-(3″,4″-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formly-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1μM), PMA (100nM) or leukotriene B₄ (LTB₄; 1μM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC₅₀ value of 0.3±0.1μM. Intracellular signaling caused by fMLP, PMA or LTB₄ were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB₄ or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC₅₀ of 1.5±0.2μM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.

Keywords: Piper philippinum; Lignan; PP-6; Neutrophil; fMLP; Superoxide anion

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity by Saskia Eckert; Peter Eyer; Nadja Herkert; Rudolf Bumm; Georg Weber; Horst Thiermann; Franz Worek (pp. 698-703).

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex^®-GS, 0.22μm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax^® and Potter–Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10μM) or HI 6 (30μM) of sarin-inhibited human muscle acetylcholinesterase (0.142±0.004min⁻¹ and 0.166±0.008min⁻¹, respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.

Keywords: Abbreviations; AChE; acetylcholinesterase (EC 3.1.1.7); AU; absorbance units; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid); HI 6; 1-[[[4-(aminocarbonyl)-pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride; Obi (obidoxime); 1,1′-(oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride; Sarin; isopropyl methylphosphonofluoridateAcetylcholinesterase; Muscle; Oxime; Paraoxon; Sarin

Hepatic alteration of tryptophan metabolism in an acute porphyria model by Sandra M. Lelli; Marta B. Mazzetti; Leonor C. San Martín de Viale (pp. 704-712).

This study focuses on the alterations suffered by the serotoninergic and kinurenergic routes of tryptophan (TRP) metabolism in liver, and their relation with gluconeogenic phosphoenolpyruvate-carboxykinase (PEPCK) blockage in experimental acute porphyria. This porphyria was induced in rats by a combined treatment of 2-allyl-2-isopropylacetamide (100, 250, 500mg/kg bw) and 3,5-dietoxicarbonil 1,4-dihydrocollidine (constant 50mg/kg bw dose).Results showed a marked dose-dependent increase of all TRP pyrrolase (TRPp) forms, active (holo, total) and inactive (apo), and a decrease in the degree of enzyme saturation by heme. Increases for holo, total, and apo-TRPp were 90, 150, and 230%, respectively, at the highest dose assayed ( H). The treatment also impaired the serotoninergic route of TRP metabolism in liver, causing a decrease in serotonin level ( H, 38%), and a concomitant enhancement in TRP content ( H, 23%).The porphyrinogenic treatment promoted a blockage in PEPCK activity ( H, 30%). This occurred in correlation to the development of porphyria, to TRPp alterations and to the production of hepatic microsomal thiobarbituric acid reactive substances. Porphyria was estimated through increases in 5-aminolevulinic acid-synthase (ALA-S) activity, ALA and porphobilinogen contents, and a decrease in ferrochelatase activity.Thus, the TRP kynurenine route was augmented whereas the serotoninergic route was reduced. PEPCK blockage could be partly attributed to quinolinate generated from TRP by the increase of TRPp activity, which would be due to the effect of porphyrinogenic drugs on TRP. The contribution of ROS to PEPCK blockage is analyzed. Likewise, the implication of these results in the control of porphyrias by glucose is discussed.

Keywords: Abbreviations; AIA; 2-allyl-2-isopropylacetamide; ALA; 5-aminolevulinic acid; ALA-S; ALA-synthase; DDC; 3,5-diethoxycarbonyl-1,4-dihydrocollidine; PCT; porphyria cutanea tarda; PEPCK; phosphoenolpyruvate-carboxykinase; PBG; porphobilinogen; Proto; protoporphyrin; ROS; reactive oxygen species; 5-HT; serotonin; TBARS; thiobarbituric acid reactive substances; TRP; tryptophan; TRPp; tryptophan pyrrolaseAcute porphyria model; Tryptophan metabolism; Tryptophan pyrrolase; Phosphoenolpyruvate-carboxykinase; 2-Allyl-2-isopropylacetamide; 3,5-Diethoxycarbonyl-1,4-dihydrocollidine

N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription and activity in human breast cancer cells by Sarah M. Jump; Jenny Kung; Richard Staub; Matthew A. Kinseth; Erin J. Cram; Larisa N. Yudina; Maria N. Preobrazhenskaya; Leonard F. Bjeldanes; Gary L. Firestone (pp. 713-724).

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, induces a G1 cell cycle arrest of human breast cancer cells. Structure–activity relationships of I3C that mediate this anti-proliferative response were investigated using synthetic and natural I3C derivatives that contain substitutions at the indole nitrogen. Nitrogen substitutions included N-alkoxy substituents of one to four carbons in length, which inhibit dehydration and the formation of the reactive indolenine. Analysis of growth and cell cycle arrest of indole-treated human breast cancer cells revealed a striking increase in efficacy of the N-alkoxy I3C derivatives that is significantly enhanced by the presence of increasing carbon lengths of the N-alkoxy substituents. Compared to I3C, the half maximal growth arrest responses occurred at 23-fold lower indole concentration for N-methoxy I3C, 50-fold lower concentration for N-ethoxy I3C, 217-fold lower concentration for N-propoxy I3C, and 470-fold lower concentration for N-butoxy I3C. At these lower concentrations, each of the N-alkoxy substituted compounds induced the characteristic I3C response in that CDK6 gene expression, CDK6 promoter activity, and CDK2 specific enzymatic activity for its retinoblastoma protein substrate were strongly down regulated. 3-Methoxymethylindole and 3-ethoxymethylindole were approximately as bioactive as I3C, whereas both tryptophol and melatonin failed to induce the cell cycle arrest, showing the importance of the C-3 hydroxy methyl substituent on the indole ring. Taken together, our study establishes the first I3C structure–activity relationship for cytostatic activities, and implicates I3C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer.

Keywords: Abbreviations; I3C; indole-3-carbinol; DIM; 3,3′-diindolylmethane; CDK; cyclin-dependent kinaseI3C; Synthetic derivatives; N; -Alkoxy constituents; Breast cancer cells; Cell cycle arrest

Biochemical mechanisms underlying the pro-apoptotic activity of 7,8-dihydroxy-4-methylcoumarin in human leukemic cells by Maria E. Riveiro; Ramiro Vazquez; Albertina Moglioni; Natalia Gomez; Alberto Baldi; Carlos Davio; Carina Shayo (pp. 725-736).

The search for new drugs requires a deep understanding of the molecular basis of drug action, being necessary the elucidation of the mechanism of action with the understanding of the relationship between structure and activity. In the present study, we evaluated the pro-apoptotic activity of 7,8-dihydroxy-4-methylcoumarin (DHMC) and its underlying mechanisms in human leukemic cells. Here, we present evidence that DHMC induced selective and concentration-dependent apoptosis in human leukemic cells. The pro-apoptotic effect of DHMC was mediated by activation of the JNKs and inhibition of the ERK1/2 and PI3K/Akt pathways, with no participation of the p38 cascade after 24h of treatment. Indeed, down-regulation of the proto-oncogene c-myc as well as induction of the cell cycle inhibitor p21 WAF1/^CIP1 through a p53 independent mechanism were observed in U-937 cells. These findings suggest that DHCM may have a potential therapeutic role in the future treatment of hematological malignancies.

Keywords: Abbreviations; DHMC; 7,8-dihydroxy-4-methylcoumarin; PBS; phosphate-buffered saline; DMSO; dimethyl sulfoxide; SDS; sodium dodecyl sulfate; TBE buffer; tris-borate–EDTA; NAC; N; -acetyl-; l; -cysteine; IC; ₅₀; inhibitory proliferation concentration 50; CC; ₅₀; cytotoxic concentration 50; HO; Hoechst 33342; PI; propidium iodideLeukemia; Hydroxycoumarins; DHMC; Apoptosis induction; Signal transduction

The reversibility of neurofilaments decline induced by 2,5-hexanedione in rat nerve tissues by Fuyong Song; Sufang Yu; Cuili Zhang; Guizhen Zhou; Qingshan Wang; Keqin Xie (pp. 737-744).

To investigate the reversibility of the neuropathy induced by 2,5-HD, adult male rats were administered at a dosage of 400mg/kg/day 2,5-HD (five times per week) for 2, 4, and 8 weeks, respectively. After stopping HD exposure, half of 8-week treated animals were allowed to naturally recover for 16 weeks. The relative levels of NF-H, NF-M, and NF-L in spinal cords and sciatic nerves of rats were determined by immunoblotting during the HD neuropathy. The results showed that NFs content in nerve tissues demonstrated a progressive decline as the intoxication continued. Furthermore, after a recovery of 16 weeks, the levels of three NF subunits in spinal cords of treated rats returned to normal while those in sciatic nerves displayed an inconsistent reversal. Among them, the level of NF-H in sciatic nerves returned to normal completely, and NF-L also showed a significant improvement, whereas NF-M did not demonstrate an obvious reversal. These findings suggest that HD-induced NFs decline is at least partially irreversible within the time frame of this study, which might be associated with the incomplete recovery of neurological dysfunctions of HD-treated rats.

Keywords: 2,5-Hexanedione; n; -Hexane; Neurofilament; Spinal cord; Sciatic nerve; Toxic axonopathy

Contributions of phosphorylation to regulation of OCTN2 uptake of carnitine are minimal in BeWo cells by Erik Rytting; Kenneth L. Audus (pp. 745-751).

Physiological functions of organic cation transporters (OCTs) in the placenta include transporting essential nutrients from the maternal to fetal circulations. OCTN2 transports carnitine with high affinity, and the transport of several drugs has also been shown to be mediated by this transporter. In this work, the role of phosphorylation and dephosphorylation mechanisms in regulating OCTN2 was investigated by observing the effects of various activators and inhibitors of kinases and phosphatases on the uptake of carnitine in BeWo cells, a human choriocarcinoma trophoblast cell line frequently used as an in vitro model of the rate-limiting barrier for maternal-fetal exchange. Preincubation with genistein resulted in significant increases in both alkaline phosphatase (ALP) activity and carnitine uptake. Levamisole, an ALP inhibitor, caused a more substantial decrease in carnitine uptake than expected from its corresponding decrease in ALP activity. It was determined that levamisole competitively inhibits carnitine uptake, with a K_i value of 1.01±0.05mM, and this effect has a greater role in decreasing carnitine uptake than any indirect effects of ALP inhibition upon OCTN2 function. Progesterone also competitively inhibited carnitine uptake ( K_i=48.6±5.0μM), but had no effect on ALP activity in BeWo cells.

Keywords: Organic cation transporters; OCTN2; Placenta; Carnitine; Phosphorylation; Levamisole

Oxidation of 5-methoxy- N, N-diisopropyltryptamine in rat liver microsomes and recombinant cytochrome P450 enzymes by Shizuo Narimatsu; Rei Yonemoto; Kazufumi Masuda; Takashi Katsu; Masato Asanuma; Tooru Kamata; Munehiro Katagi; Hitoshi Tsuchihashi; Takuya Kumamoto; Tsutomu Ishikawa; Shinsaku Naito; Shigeru Yamano; Nobumitsu Hanioka (pp. 752-760).

The oxidative metabolism of 5-methoxy- N, N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy- N, N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377–85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with β-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.

Keywords: 5-Methoxy-; N; ,; N; -diisopropyltryptamine; Rat; O; -Demethylation; N; -Deisopropylation; 6-Hydroxylation; CYP2C11; CYP2D2; CYP3A2; CYP1A1

Differential regulation of cell death in head and neck cell carcinoma through alteration of cholesterol levels in lipid rafts microdomains by Clara Bionda; Anne Athias; Delphine Poncet; Gersende Alphonse; Amel Guezguez; Philippe Gambert; Claire Rodriguez-Lafrasse; Dominique Ardail (pp. 761-772).

Lipid rafts are cholesterol-enriched microdomains in the plasma membrane. They act as molecular platforms that spatially organize membrane receptor molecules and are involved in the transduction of various signaling pathways. We recently reported that in the radiosensitive squamous cell carcinoma SCC61 line, γ-irradiation results in a rearrangement of the plasma membrane rafts and signaling platforms leading to radiation-induced apoptosis in a ceramide-dependent pathway. By contrast, this reorganization was found to be defective in the radioresistant counterpart cell line, SQ20B. As the cholesterol content of lipid rafts is two times higher in SQ20B compared with SCC61 cells, we investigated the modulation of these microdomains using methyl-beta-cyclodextrin (MβCDX), a widely used cholesterol-depleting agent, in order to disrupt raft organization in both cells. Here, we report that MβCDX treatment resulted in the triggering of apoptosis in SCC61 cells involving mitochondrial events and associated with the clustering of Fas, the formation of Fas–FADD complexes and the cleavage of procaspase 8. The ligand-independent activation of this death receptor was totally absent in SQ20B cells, which remained resistant to MβCDX-triggered apoptosis. However, treatment of SQ20B with MβCDX resulted in a ligand-independent activation of the epidermal growth factor receptor (EGFR) survival pathway, as evidenced by an increased tyrosine phosphorylation of EGFR. Taken altogether, our results indicate that lipid raft integrity is intimately involved in the triggering of apoptotic cell death and/or survival pathways in head and neck carcinoma cells.

Keywords: Methyl-beta-cyclodextrine; Lipid rafts; Cholesterol; Apoptosis; Head and neck squamous cell carcinoma; Fas; EGF receptor

Protective effect of N-acetylcysteine against radiation induced DNA damage and hepatic toxicity in rats by Heba H. Mansour; Hafez F. Hafez; Nadia M. Fahmy; Nemat Hanafi (pp. 773-780).

The present study was designed to evaluate the radioprotective effect of N- acetylcysteine (NAC) on γ-radiation induced toxicity in hepatic tissue in rat. The cellular changes were estimated using malondialdehyde (MDA, an index of lipid peroxidation), superoxide dismutase (SOD), glutathione peroxidase (GSHPx), reduced glutathione (GSH), and total nitrate/nitrite (NO(x)) as markers of hepatic oxidative stress in rats following γ-irradiation. The DNA damage was determined by agarose gel electrophoresis. To achieve the ultimate goal of this study, 40 adult rats were randomly divided into 4 groups of 10 animals each. Group I was injected intraperitoneally with saline solution for 7 consecutive days and served as control group. Group II was irradiated with a single dose of 6Gy γ-radiation. Group III was daily injected with NAC (1g/kg, i.p.) for 7 consecutive days. Group IV received a daily i.p. injection of NAC (1g/kg, i.p.) for 7 consecutive days and 1h after the last dose, rats were irradiated with a single dose (6Gy) γ-radiation. The animals were sacrified after 24h. DNA damage was observed in tissue after total body irradiation with a single dose of 6Gy. Malondialdehyde and total nitrate/nitrite were increased significantly whereas the levels of GSH and antioxidant enzymes were significantly decreased in γ-irradiated group. Pretreatment with NAC showed a significant decrease in the levels of MDA, NO(x) and DNA damage. The antioxidant enzymes increased significantly along with the levels of GSH. Moreover, histopathological examination of liver tissues confirmed the biochemical data. Thus, our results show that pretreatment with N-acetylcysteine offers protection against γ-radiation induced cellular damage.

Keywords: N; -Acetylcysteine; γ-Radiation; Antioxidant; Nitric oxide and DNA damage

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Biochemical Pharmacology (v.75, #3)

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Biochemical Pharmacology (v.75, #3)