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Biochemical Pharmacology (v.72, #10)

Editorial Advisory Board (pp. iii).

Recent clinical failures in Parkinson's disease with apoptosis inhibitors underline the need for a paradigm shift in drug discovery for neurodegenerative diseases by Peter Waldmeier; Donna Bozyczko-Coyne; Michael Williams; Jeffry L. Vaught (pp. 1197-1206).

Understanding the mechanisms of neuronal death in concert with the identification of drugable molecular targets key to this process has held great promise for the development of novel chemical entities (NCEs) to halt neurodegenerative disease progression. Two key targets involved in the apoptotic process identified over the past decade include the mixed lineage kinase (MLK) family and glyceraldehyde phosphate dehydrogenase (GAPDH). Two NCEs, CEP-1347 and TCH346, directed against these respective targets have progressed to the clinic. For each, robust neuroprotective activity was demonstrated in multiple in vitro and in vivo models of neuronal cell death, but neither NCE proved effective Parkinson's disease (PD) patients. These recent clinical failures require a reassessment of both the relevance of apoptosis to neurodegenerative disease etiology and the available animal models used to prioritize NCEs for advancement to the clinic in this area.

Keywords: Apoptosis; Necrosis; Parkinson's disease; CEP-1347; TCH346; Animal models; Clinical trial failure

The HSP90 and DNA topoisomerase VI inhibitor radicicol also inhibits human type II DNA topoisomerase by Danièle Gadelle; Marc Graille; Patrick Forterre (pp. 1207-1216).

Radicicol derivatives are currently investigated as promising antitumoral drugs because they inhibit the activity of the molecular chaperone heat shock protein (HSP90), causing the destabilization and eventual degradation of HSP90 client proteins that are often associated with tumor cells. These drugs interact with the ATP-binding site of HSP90 which is characterized by a structural element known as the Bergerat fold, also present in type II DNA topoisomerases (Topo II). We have previously shown that radicicol inhibits archaeal DNA topoisomerase VI, the prototype of Topo II of the B family (present in archaea, some bacteria and all the plants sequenced so far). We show here that radicicol also inhibits the human Topo II, a member of the A family (comprising the eukaryotic Topo II, bacterial gyrase, Topo IV and viral Topo II), which is a major target for antitumoral drugs. In addition, radicicol prevents in vitro induction of DNA cleavage by human Topo II in the presence of the antitumoral drug etoposide. The finding that radicicol can inhibit at least two different antitumoral drug targets in human, and interferes with drugs currently used in cancer treatment, could have implications in cancer therapy.

Keywords: Radicicol; Bergerat fold; GHKL family; Type II DNA topoisomerase; HSP90; Etoposide

Fluorinated Cpd 5, a pure arylating K-vitamin derivative, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating MAPK by Siddhartha Kar; Meifang Wang; Seung Wook Ham; Brian I. Carr (pp. 1217-1227).

We previously synthesized several K-vitamin derivatives, which are potent growth inhibitors of human tumor cells, including Hep3B human hepatoma cells. Among these, Cpd 5 was the most potent. However, being a quinone derivative, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a fluorinated derivative of Cpd 5, F-Cpd 5. The calculated reduction potential of F-Cpd 5 was much higher than that for Cpd 5 and it was not predicted to generate ROS. This was supported by our observation that F-Cpd 5 generated significantly lower ROS than Cpd 5. F-Cpd 5 was three times more potent than Cpd 5 in inhibiting Hep3B cell growth. Interestingly, under identical culture conditions, F-Cpd 5 inhibited mitogen-induced DNA synthesis in normal rat hepatocytes 12-fold less potently than Hep3B cells. F-Cpd 5 was found to induce caspase-3 cleavage and nuclear DNA laddering, evidences for apoptosis. It preferentially inhibited the activities of the cell cycle controlling phosphatases Cdc25A and Cdc25B, by binding to their catalytic cysteines. Consequently, inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 were induced. F-Cpd 5 also induced phosphorylation of the MAPK proteins ERK1/2, JNK1/2 and p38 in Hep3B cells and the MAPK inhibitors (U0126, JNKI-II, and SB 203580) antagonized its growth inhibition. F-Cpd 5 inhibited the action of cytosolic ERK phosphatase activity, which likely caused the ERK phosphorylation. F-Cpd 5 thus differentially inhibited growth of normal and tumor cells by preferentially inhibiting the actions of Cdc25A and Cdc25B phosphatases and inducing MAPK phosphorylation.

Keywords: Abbreviations; PTP; protein tyrosine phosphatase; ERK; extracellular signal related kinase; pERK; dual phosphorylated ERK; MAPK; mitogen activated protein kinase; EGF; epidermal growth factor; F-Cpd 5; fluorinated Cpd 5Liver cancer; Protein phosphatase; K-vitamin derivative; Cell cycle inhibition; Cdc25; MAPK

Synergistic induction of apoptosis by sulindac and arsenic trioxide in human lung cancer A549 cells via reactive oxygen species-dependent down-regulation of survivin by Hyeon-Ok Jin; Su-Im Yoon; Sung-Keum Seo; Hyung-Chahn Lee; Sang-Hyeok Woo; Doo-Hyun Yoo; Su-Jae Lee; Tae-Boo Choe; Sungkwan An; Tae-Jong Kwon; Jong-Il Kim; Myung-Jin Park; Seok-Il Hong; In-Chul Park; Chang-Hun Rhee (pp. 1228-1236).

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, may be a good target for cancer therapy because it is expressed in a variety of human tumors but not in differentiated adult tissues. In the present study, we show that a combination of sulindac and arsenic trioxide (ATO) induces more extensive apoptosis than either drug alone in A549 human non-small cell lung carcinoma (NSCLC) cells. Treatment with sulindac/ATO reduced the expression of survivin and promoted major apoptotic signaling events, namely, collapse of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. Combined sulindac/ATO treatment did not significantly affect the levels of other members of the IAP family (XIAP, cIAP1 and cIAP2), indicating that the effects were specific to survivin. In addition, sulindac/ATO treatment induced the production of reactive oxygen species and the antioxidant N-acetyl-l-cysteine blocked the down-regulation of survivin and induction of apoptotic signaling by the combination of sulindac and ATO. Combined sulindac/ATO treatment also activated p53 expression, and inhibition of p53 expression by small interfering RNA (siRNA) prevented sulindac/ATO-induced down-regulation of survivin, suggesting that survivin expression is negatively regulated by p53. Overexpression of survivin reduced sulindac/ATO-induced apoptosis in A549 cells and reduction of survivin levels by siRNA sensitized the cells to sulindac/ATO-induced cell death. These results demonstrate that, in A549 human NSCLC cells, sulindac/ATO-induced apoptosis is mediated by the reactive oxygen species-dependent down-regulation of survivin.

Keywords: Apoptosis; Arsenic trioxide; Lung cancer; NSAIDs; Survivin

Basis for dosing time-dependent change in the anti-tumor effect of imatinib in mice by Hiroo Nakagawa; Takako Takiguchi; Mariko Nakamura; Atsuko Furuyama; Satoru Koyanagi; Hironori Aramaki; Shun Higuchi; Shigehiro Ohdo (pp. 1237-1245).

Because a variety of receptor tyrosine kinases are involved in the mechanism of tumor progression, the development of a clinically useful tyrosine kinase inhibitor is expected as a therapeutic agent for the treatment of malignant cancers. Imatinib mesylate, known as Gleevec or STI-571, is a molecule that inhibits the function of various receptors with tyrosine kinase activity, such as Abl, the bcr–abl chimeric product, KIT, and platelet-derived growth factor (PDGF) receptors. In this study, we investigated the influence of dosing time on the ability of imatinib to inhibit tumor growth in mice. Tumor-bearing mice were housed under standardized light/dark cycle conditions (lights on at 07:00h, off at 19:00h) with food and water ad libitum. The growth of tumor cells implanted in mice was more severely inhibited by the administration of imatinib (50mg/kg, i.p.) in the early light phase than when it was administered in the early dark phase. The dosing time-dependency of anti-tumor effects was parallel to that of imatinib-induced anti-angiogenic effect. The inhibitory effect of imatinib on tyrosine kinase activity of PDGF receptors, but not of KIT and Abl, varied according to its administration time. The dosing time-dependency of imatinib-induced inhibition of PDGF receptor activity was closely related to that of its anti-tumor effects. Our results suggest that the anti-tumor efficacy of imatinib is enhanced by administering the drug when PDGF receptor activity was increased. The potent therapeutic efficacy of the drug could be expected by optimizing the dosing schedule.

Keywords: Abbreviations; PDGF; platelet-derived growth factor; ERK; extracellular signal-regulated kinases; MAP; mitogen-activated protein kinase; PI3K; phosphoinositol-3-kinaseImatinib; Tyrosine kinase; PDGF receptor; Cancer therapy; Circadian rhythm; Chronopharmacotherapy

Inhibition of phosphatidylinositol 3-kinase-mediated glucose metabolism coincides with resveratrol-induced cell cycle arrest in human diffuse large B-cell lymphomas by Anthony C. Faber; Fay J. Dufort; Derek Blair; Dean Wagner; Mary F. Roberts; Thomas C. Chiles (pp. 1246-1256).

An abnormally high rate of aerobic glycolysis is characteristic of many transformed cells. Here we report the polyphenolic compound, resveratrol, inhibited phosphatidylinositol 3-kinase (PI-3K) signaling and glucose metabolism, coinciding with cell-cycle arrest, in germinal center (GC)-like LY1 and LY18 human diffuse large B-cell lymphomas (DLBCLs). Specifically, resveratrol inhibited the phosphorylation of Akt, p70 S6K, and S6 ribosomal protein on activation residues. Biochemical analyses and nuclear magnetic resonance spectroscopy identified glycolysis as the primary glucose catabolic pathway in LY18 cells. Treatment with the glycolytic inhibitor 2-deoxy-d-glucose, resulted in accumulation of LY18 cells in G₀/G₁-phase, underscoring the biological significance of glycolysis in growth. Glycolytic flux was inhibited by the PI-3K inhibitor LY294002, suggesting a requirement for PI-3K activity in glucose catabolism. Importantly, resveratrol treatment resulted in inhibition of glycolysis. Decreased glycolytic flux corresponded to a parallel reduction in the expression of several mRNAs encoding rate-limiting glycolytic enzymes. These results are the first to identify as a mechanism underlying resveratrol-induced growth arrest, the inhibition of glucose catabolism by the glycolytic pathway. Taken together, these results raise the possibility that inhibition of signaling and metabolic pathways that control glycolysis might be effective in therapy of DLBCLs.

Keywords: Resveratrol; Phosphatidylinositol 3-kinase; Cell cycle; Glycolysis; Diffuse large B cell lymphoma

Gene expression profiling in R-flurbiprofen-treated prostate cancer: R-Flurbiprofen regulates prostate stem cell antigen through activation of AKT kinase by Marina Zemskova; William Wechter; Svetlana Bashkirova; Chien-Shing Chen; Robert Reiter; Michael B. Lilly (pp. 1257-1267).

We have used gene expression profiling to characterize genes regulated by the anti-tumor non-steroidal anti-inflammatory drug (NSAID)-like agent R-flurbiprofen (RFB) in murine TRAMP prostate cancer. Mice with spontaneous, palpable tumors were treated with RFB 25mg/(kgd)×7d orally, or vehicle only. RNA was then extracted from tumor tissue and used for microarray analysis with Affymetrix chips. Fifty-eight genes were reproducibly regulated by RFB treatment. One of the most highly up-regulated genes was prostate stem cell antigen ( psca). We used TRAMP C1 murine prostate cancer cells to examine potential mechanisms through which RFB could regulate psca. RFB induced dose-dependent expression of PSCA protein, and activity of the psca promoter, in TRAMP C1 cells in culture. Increased psca promoter activity was also seen following treatment of cells with sulindac sulfone, another NSAID-like agent, but not with celecoxib treatment. RFB activation of the psca promoter could be attenuated by co-transfection of dominant-negative akt and h-ras constructs, but not by dominant-negative mek1 plasmids. Immunoblotting revealed that RFB increased expression of phosphorylated AKT at concentrations that stimulated psca promoter activity, and that increased PSCA protein expression. In addition, RFB-dependent up-regulation of PSCA protein expression could be blocked by AKT inhibitors. These data demonstrate that RFB, and possibly other NSAID-like analogs, can increase expression of the psca gene both in vivo and in culture. They further suggest the utility of combining RFB with AKT inhibitors or with monoclonal antibodies targeting PSCA protein, for treatment or prevention of prostate cancer.

Keywords: Prostate cancer; Microarray; NSAID; R; -Flurbiprofen; PSCA; TRAMP; AKT

Quercetin, a flavonoid, inhibits proliferation and increases osteogenic differentiation in human adipose stromal cells by Yeon Jeong Kim; Yong Chan Bae; Kuen Taek Suh; Jin Sup Jung (pp. 1268-1278).

Flavonoids, which have been detected in a variety of foods, have been repeatedly reported to affect bone metabolism. However, the effects of flavonoids on osteoblastogenesis remain a matter of some controversy. In this study, the effects of quercetin on the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) were determined. Quercetin was found to increase osteogenic differentiation in a dose-dependent manner. Other flavonoids, chrysin and kaempferol, were also shown to increase the osteogenic differentiation of hADSC, but this stimulatory effect was weaker than that associated with quercetin. Quercetin pretreatment administered prior to the induction of differentiation also exerted stimulatory effects on the osteogenic differentiation of hADSC. RT-PCR and real time PCR analysis showed that quercetin treatment induced an increase in the expression of osteopontin, BMP2, alkaline phosphatase and Runx2. Quercetin inhibited the proliferation of hADSC, but did not affect their survival. The pretreatment of quercetin increased ERK phosphorylation during osteogenic differentiation, although it did not increase ERK activity in control culture condition. ICI182780, an specific estrogen receptor antagonist, failed to inhibit the effects of quercetin on osteogenic differentiation. Quercetin-pretreated hADSC showed better bone regenerating ability in skull defect model of nude mice than naive cells. Our findings indicate that quercetin enhances osteogenic differentiation via an independent mechanism from estrogen receptor (ER) activation, and prove useful for in vivo bone engineering, using human mesencymal stem cells (hMSC).

Keywords: Quercetin; Osteogenic differentiation; Human adipose stromal cells; Proliferation; ERK; Bone regeneration

PTP1B-dependent insulin receptor phosphorylation/residency in the endocytic recycling compartment of CHO-IR cells by Wanda A. Cromlish; Man Tang; Robert Kyskan; Linda Tran; Brian P. Kennedy (pp. 1279-1292).

Insulin binds to the α subunit of the insulin receptor (IR) on the cell surface. The insulin–IR complex is subsequently internalized and trafficked within the cell. Endocytosed receptors, devoid of insulin, recycle back to the plasma membrane through the endocytic recycling compartment (ERC). Using a high content screening system, we investigate the intracellular trafficking of the IR and its phosphorylation state, within the ERC, in response to protein tyrosine phosphatase-1B (PTP1B) inhibition. Insulin stimulates, in a time- and dose-dependent manner, the accumulation of phosphorylated IR (pY^{1158,1162,1163} IR) in the ERC of CHO-IR cells. Treatment of CHO-IR cells with PTP1B-specific inhibitors or siRNA leads to dose-dependent increases in IR residency and phosphorylation within the ERC. The results also demonstrate that PTP1B redistributes within CHO-IR cells upon insulin challenge. The established system will allow for efficient screening of candidate inhibitors for the modulation of PTP1B activity.

Keywords: Abbreviations; HCS; high content screen; CHO; Chinese hamster ovary; CHO-IR; Chinese hamster ovary cells overexpressing the human insulin receptor; ERC; endocytic recycling compartment; PTP1B; protein tyrosine phosphatase-1B; IR; insulin receptor; pY-IR or pY; ^{1158,1162,1163}; IR; insulin receptor phosphorylated on tyrosines 1158, 1162, 1163; FBS; fetal bovine serum; TBS; tris-buffered saline; HBSS; Hanks buffered saline solution; FITC; fluorescein-5-isothiocyanateInsulin signalling; Receptor internalization; Protein tyrosine phosphatase-1B; CHO-IR cells; Endocytic recycling compartment; High content screening

Acacetin suppressed LPS-induced up-expression of iNOS and COX-2 in murine macrophages and TPA-induced tumor promotion in mice by Min-Hsiung Pan; Ching-Shu Lai; Ying-Jan Wang; Chi-Tang Ho (pp. 1293-1303).

Acacetin (5,7-dihydroxy-4′-methoxyflavone), a flavonoid compound, has anti-peroxidative and anti-inflammatory effects. In this study, we investigated the inhibitory effects of acacetin and a related compound, wogonin, on the induction of NO synthase (NOS) and COX-2 in RAW 264.7 cells activated with lipopolysaccharide (LPS). Acacetin markedly and actively inhibited the transcriptional activation of iNOS and COX-2. Western blotting, reverse transcription-polymerase chain reaction (PCR), and real-time PCR analyses demonstrated that acacetin significantly blocked protein and mRNA expression of iNOS and COX-2 in LPS-inducted macrophages. Treatment with acacetin reduced translocation of nuclear factor-κB (NFκB) subunit and the dependent transcriptional activity of NFκB. The activation of NFκB was inhibited by prevention of the degradation of inhibitor κB (IκB). Furthermore, acacetin inhibited LPS-induced phosphorylation as well as degradation of IκBα. We further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) in LPS-induced macrophages. We found that acacetin also inhibited LPS-induced activation of PI3K/Akt and p44/42, but not p38 MAPK. After initiation of 7,12-dimethlybene[ a]anthracene (DMBA), applying acacentin topically before each 12- O-tetradecanoylphorbol 13-acetat (TPA) treatment was found to reduce the number of papillomas at 20 weeks. Taken together, these results show that acacetin down regulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NFκB by interfering with the activation PI3K/Akt/IKK and MAPK, suggesting that acacetin is a functionally novel agent capable of preventing inflammation-associated tumorigenesis.

Keywords: Abbreviations; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; NO; nitric oxide; COX-2; cycoloxygenase-2; PGE; ₂; prostaglandin E; ₂; MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; iκB; inhibitor κB; NF-κB; nuclear factor-κB; MAPK; mitogen-activated protien kinaseAcacetin; Inducible NO synthesis (iNOS); NFκB; RAW 264.7 monocyte/macrophages; 12-; O; -Tetradecanoylphorbol 13-acetat (TPA); 7,12-Dimethylbenz[; a; ]anthracene (DMBA); Lipopolysaccharide (LPS); Mitogen-activated protein (MAPK); Phosphatidylinositiol 3-kinase (PI3K/Akt); Cyclooxygenase-2 (COX-2)

Therapeutic and preventive properties of quercetin in experimental arthritis correlate with decreased macrophage inflammatory mediators by Maria Mamani-Matsuda; Tina Kauss; Abir Al-Kharrat; Jérôme Rambert; Fawaz Fawaz; Denis Thiolat; Daniel Moynet; Sara Coves; Denis Malvy; M. Djavad Mossalayi (pp. 1304-1310).

Pentahydroxyflavone dihydrate, quercetin (QU) is one of common flavonols biosynthesized by plants and has been suggested to modulate inflammatory responses in various models. In the present study, we investigated in vivo effects of oral or intra-cutaneous QU in chronic rat adjuvant-induced arthritis (AA). Growth delay and arthritic scores were evaluated daily after AA induction in Lewis rats. Oral administration of QU (5× 160mg/kg) to arthritic rats resulted in a clear decrease of clinical signs compared to untreated controls. Intra-cutaneous injections of lower doses (5× 60mg/kg) of QU gave similar anti-arthritic effects, while 5× 30mg/kg concentrations were inefficient in this respect. Finally, injection of relatively low QU doses (5× 30mg/kg) prior to AA induction significantly reduced arthritis signs. As QU was suggested to inhibit macrophage-derived cytokines and nitric oxide (NO), we then analyzed macrophage response ex vivo. Anti-arthritic effects of QU correlated with significant decrease of inflammatory mediators produced by peritoneal macrophages, ex vivo and in vitro. These data indicate that QU is a potential anti-inflammatory therapeutic and preventive agent targeting the inflammatory response of macrophages.

Keywords: Abbreviations; AA; adjuvant-induced arthritis; COX; cyclooxygenase; I.C.; intra-cutaneous; IFN; interferon; NOS; nitric oxide synthase; QU; quercetin; RA; rheumatoid arthritis; TNF; tumor necrosis factorInflammation; Quercetin; Rheumatoid arthritis; Macrophage

Inhibition of NF-κB activation through targeting IκB kinase by celastrol, a quinone methide triterpenoid by Jeong-Hyung Lee; Tae Hyeon Koo; Hyunkyung Yoon; Haeng Sun Jung; Hui Zi Jin; Kyeong Lee; Young-Soo Hong; Jung Joon Lee (pp. 1311-1321).

Celastrol, a quinone methide triterpenoid, was isolated as an inhibitor of NF-κB from Celastrus orbiculatus. This compound dose-dependently inhibited a variety of stimuli-induced NF-κB-regulated gene expression and the DNA-binding of NF-κB in different cell lines without affecting DNA-binding activity of AP-1. Preincubation of celastrol completely blocked the LPS-, TNF-α-, or PMA-induced degradation and phosphorylation of IκBα. Importantly, celastrol inhibited IKK activity and the constitutively active IKKβ activity in a dose-dependent manner without either affecting the NF-κB activation induced by RelA over-expression or directly suppressing the DNA-binding of activated NF-κB. However, mutation of cysteine 179 in the activation loop of IKKβ abolished sensitivity towards to celastrol, suggesting that celastrol suppressed the NF-κB activation by targeting cysteine 179 in the IKK. To verify that celastrol is a NF-κB inhibitor, we investigated its effect on some NF-κB target genes expressions. Celastrol prevented not only LPS-induced mRNA expression of iNOS and TNF-α, but also TNF-α-induced Bfl-1/A1 expression, a prosurvival Bcl-2 homologue. Consistent with these results, celastrol significantly suppressed the production of NO and TNF-α in LPS-stimulated RAW264.7 cells, and increased the cytotoxicity of TNF-α in HT-1080 cells. We also demonstrated that celastrol showed anti-inflammatory and anti-tumor activities in animal models. Taken together, this study extends our understanding on the molecular mechanisms underlying the anti-inflammatory and anti-cancer activities of celastrol and celastrol-containing medicinal plant, which would be a valuable candidate for the intervention of NF-κB-dependent pathological conditions.

Keywords: Abbreviations; NF-κB; nuclear factor κB; AP-1; activator protein-1; LPS; lipopolysaccharide; TNF-α; tumor necrosis factor-α; PMA; phorbol myristyl acetate; MEKK-1; mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1; IKK; IκB kinase; EMSA; electrophoretic mobility shift assay; COX-2; cyclooxygenase-2; PGE; ₂; prostaglandin E; ₂; iNOS; inducible nitric oxide synthase; NO; nitric oxide; MPO; myeloperoxidase; DTT; dithiothreitolCelastrol; Quinone methide triterpenoid; NF-κB; IκB kinase; Anti-inflammatory and anti-tumor activity

2′,4′,6′-Tris(methoxymethoxy) chalcone attenuates hepatic stellate cell proliferation by a heme oxygenase-dependent pathway by Sung Hee Lee; Geom Seog Seo; Hak Sung Kim; Sun Wook Woo; Geonil Ko; Dong Hwan Sohn (pp. 1322-1333).

Proliferation of hepatic stellate cells (HSCs) is central for the development of fibrosis during liver injury. We have shown previously that butein (3,4,2′,4′-tetrahydroxychalcone) suppresses myofibroblastic differentiation of rat HSCs. Our aim in this study was to determine whether a new synthetic chalcone derivative, 2′,4′,6′-tris(methoxymethoxy) chalcone (TMMC) inhibits HSC proliferation induced by serum- or platelet-derived growth factor (PDGF). TMMC significantly inhibited growth factor-induced HSC proliferation. The inhibition of PDGF-induced proliferation by TMMC was associated with the phosphatidylinositol 3-kinase-Akt-p70^S6K pathways. TMMC induced the expression of heme oxygenase 1 (HO-1) in HSCs. Using the chemical inhibitor tin protoporphyrin, we also found that the inhibitory action of TMMC on PDGF-induced proliferation is mediated by HO-1. Glutathione (GSH) depletion produced by TMMC activated extracellular signal-regulated kinase (ERK), which led to c-Fos expression and transactivation of activator protein 1 (AP-1) and HO-1 gene expression in the HSCs. These results demonstrate that TMMC preferentially activates ERK and that this activation leads to the transcriptional activation of AP-1 and consequently to HO-1 expression. HO-1 expression might be responsible for the antiproliferative effect of TMMC on HSCs.

Keywords: Abbreviations; AP-1; activator protein-1; DEM; diethyl maleate; ERK; extracellular signal-regulated kinase; HSC; hepatic stellate cell; HO-1; heme oxygenase-1; MAPK; mitogen-activated protein kinase; MEK; mitogen-activated protein kinase kinase; PI3K; phosphatidylinositol 3-kinase; SnPP; tin protoporphyrin; TMMC; 2′,4′,6′-tris(methoxymethoxy) chalcone2′,4′,6′-Tris(methoxymethoxy) chalcone; GSH; ERK; AP-1; Hepatic stellate cell

Radiocontrast media cause dephosphorylation of Akt and downstream signaling targets in human renal proximal tubular cells by Michele Andreucci; Giorgio Fuiano; Pierangela Presta; Pasquale Esposito; Teresa Faga; Vincenzo Bisesti; Alfredo Procino; Vincenzo Altieri; Carmela Tozzo; Bruno Memoli; Ashour Michael (pp. 1334-1342).

Radiocontrast medium induced nephrotoxicity is a major clinical problem. There is considerable interest in reducing the incidence of acute renal failure due to the use of radiocontrast media (RCM). Reduction of renal blood flow and direct toxic effect on renal tubular epithelial cells have been postulated as major causes of RCM nephropathy. Understanding the molecular mechanisms by which RCM cause cell damage may allow the development of pharmacological therapy to prevent their nephrotoxicity. In this work we have investigated the signaling pathways that may be affected by RCM.The incubation of human renal tubular proximal cells with sodium diatrizoate, iopromide and iomeprol caused a marked dephosphorylation of the kinase Akt on Ser473 within 5min of incubation. RCM also caused a decrease in cell viability, which was substantially alleviated by transfecting the cells with a constitutively active form of Akt. Further downstream targets of Akt, including the Forkhead family of transcription factors FKHR and FKHRL1, were also dephosphorylated by RCM at Thr24 and Thr32, respectively. The P70S6 kinase was also dephosphorylated at Thr389 and Ser371 by RCM. However there was a more dramatic decrease in phosphorylation of the phosphorylated form of mammalian target of rapamycin (mTOR) and of the extracellular-signal regulated kinases (ERK) 1/2 caused by sodium diatrizoate than by iopromide.These results demonstrate the effect of RCM on some intracellular signaling pathways that may allow understanding of the mechanism of their toxicity and may allow the development of strategies to overcome their adverse effects.

Keywords: Kidney; Cell survival; Nephrotoxicity; Kinase; Signaling; Tubular cells

Protective effect of monosialoganglioside GM1 against chemically induced apoptosis through targeting of mitochondrial function and iron transport by Morgane Gorria; Laurence Huc; Odile Sergent; Amélie Rebillard; François Gaboriau; Marie-Thérèse Dimanche-Boitrel; Dominique Lagadic-Gossmann (pp. 1343-1353).

Exogenous treatment with monosialoganglioside GM1 has been described to afford protection against different apoptotic insults. However, the underlying mechanisms remain to be determined. In this study, we focused on the effect of GM1 on the apoptotic cascade induced by benzo[ a]pyrene (B[ a]P) in rat hepatic F258 epithelial cells. We first demonstrated that a co-treatment with GM1 (80μM) reduced B[ a]P (50nM)-induced apoptosis as evidenced by a decrease of both cell population exhibiting nuclear fragmentation and caspase 3 cleavage and activity. We next showed that the p53 phosphorylation and nuclear translocation as well as the intracellular alkalinization related to Na⁺/H⁺ exchanger 1 (NHE1) activation, two early events of the apoptosis induced by B[ a]P, were not inhibited by GM1. In contrast, the late mitochondria-dependent acidification elicited by B[ a]P was inhibited by GM1 co-treatment, and an inhibition of the oxidative stress was also observed. Because GM1 has been shown to reduce the low-molecular weight iron content related to ethanol-induced oxidative stress, we finally investigated the involvement of iron under our conditions. Using the two iron chelators deferiprone and desferrioxamine, we clearly showed that iron played an important role in B[ a]P-induced apoptosis in F258 cells, and that B[ a]P-treatment resulted in a significant GM1-sensitive increase in⁵⁵Fe uptake. In conclusion, our results indicate that exogenous GM1 partly prevents B[ a]P-induced apoptosis by interfering with mitochondria-related intracellular acidification and iron transport.

Keywords: Abbreviations; B[; a; ]P; benzo[; a; ]pyrene; CYP1; cytochrome P450 1; DMSO; dimethyl sulfoxide; GM1; monosialoganglioside GM1; NHE1; Na; ⁺; /H; ⁺; exchanger 1; pH; _i; intracellular pH; ROS; reactive oxygen speciesBenzo[; a; ]pyrene; Apoptosis; GM1; Intracellular pH; Iron; Reactive oxygen species; Lipid peroxidation; Mitochondria

Erratum to "GLUCOSE UTILIZATION IS SUPPRESSED IN THE GUT OF INSULIN-RESISTANT HIGH FAT-FED RATS AND IS RESTORED BY METFORMIN" (pp. 1354-1354).

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Biochemical Pharmacology (v.72, #10)

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Biochemical Pharmacology (v.72, #10)