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Biochemical Pharmacology (v.73, #9)

Editorial Advisory Board (pp. iii).

Redox in redux: Emergent roles for glutathione S-transferase P (GSTP) in regulation of cell signaling and S-glutathionylation by Kenneth D. Tew (pp. 1257-1269).

Glutathione (GSH) provides a major source of thiol homeostasis critical to the maintenance of a reduced cellular environment that is conducive to cell survival. Mammals have accumulated a significant cadre of sulfur containing proteins, the interactive significance of which has become clear in recent times. Glutathione transferases (GST) are prevalent in eukaryotes and have been ascribed catalytic functions that involve detoxification of electrophiles through thioether bond formation with the cysteine thiol of GSH. The neutralizing impact of these reactions on products of reactive oxygen has contributed to the significant evolutionary conservation and adaptive functional redundancy of the multifaceted GSH system. Amongst the GSTs, GSTP has been implicated in tumorigenesis and in anticancer drug resistance. Emerging studies indicate that GSTP has ligand binding properties and contributes in the regulation of signaling kinases through direct protein:protein interactions. Furthermore, S-glutathionylation is a post-translational modification of low p K_a cysteine residues in target proteins. The forward rate of the S-glutathionylation reaction can be influenced by GSTP, whereas the reverse rate is affected by a number of redox sensitive proteins including glutaredoxin, thioredoxin and sulfiredoxin. The functional importance of these reactions in governing how cells respond to oxidative or nitrosative stress exemplifies the broad importance of GSH/GST homeostasis in conditions such as cancer, ageing and neurodegenerative diseases. GSTP has also provided a platform for therapeutic drug development where some agents have completed preclinical testing and are in clinical trial for the management of cancer.

Keywords: Glutathione; S; -Glutathionylation; Glutathione; S; -transferase; Oxidative and nitrosative stress; Cancer; C–NH; ₂; terminal jun kinase

Differences in the cellular response and signaling pathways of cisplatin and BBR3464 ([{ trans-PtCl(NH₃)₂}₂ μ-( trans-Pt(NH₃)₂(H₂N(CH₂)₆-NH₂)₂)]⁴⁺) influenced by copper homeostasis by Peyman Kabolizadeh; John Ryan; Nicholas Farrell (pp. 1270-1279).

[{ trans-PtCl(NH₃)₂}₂ μ-( trans-Pt(NH₃)₂(H₂N(CH₂)₆-NH₂)₂)]⁴⁺ (BBR3464) is a cationic trinuclear platinum drug that is being evaluated in phase II clinical trials for treatment of lung and ovarian cancers. The structure and DNA binding profile of BBR3464 is different from drugs commonly used clinically. It is of great interest to evaluate the difference between the mechanisms of uptake employed by BBR3464 and cisplatin (c-DDP), as altered uptake may explain chemoresistance. Using transfected cell lines, we show that both c-DDP and BBR3464 use the copper transporter hCTR1 to enter cells and to a lesser extent, the ATP7B transporter to exit cells. Copper influenced c-DDP and BBR3464 uptake similarly; it increased the c-DDP and BBR3464 uptake in ovarian (A2780) and colorectal (HCT116) carcinoma cell lines as detected by ICP-OES. However, the effects of copper on c-DDP- and BBR3464-mediated cytotoxicity differed. Copper decreased c-DDP-induced apoptosis, caspase-3/7 activation, p53 induction and PARP cleavage in cancer cell lines. In contrast, copper increased BBR3464-induced apoptosis, and had little effect on caspase activation, PARP cleavage, and p53 induction. It was concluded that BBR3464 employs mechanisms of intracellular action distinct from c-DDP. Although these drugs use the same cellular transporters (hCTR1 and ATP7B) for influx and efflux, downstream effects are different for the two drugs. These experiments illustrate fundamental differences in the mechanisms of action between cisplatin and the novel Pt-based drug BBR3464.

Keywords: Platinum; Copper; hCTR1; ATP7B; Chemotherapy; Apoptosis

Efficacy of increasing the therapeutic index of irinotecan, plasma and tissue selenium concentrations is methylselenocysteine dose dependent by Rami G. Azrak; Shousong Cao; Lakshmi Pendyala; Farukh A. Durrani; Marwan Fakih; Gerald F. Combs Jr.; Joshua Prey; Patrick F. Smith; Youcef M. Rustum (pp. 1280-1287).

This study was designed to understand the basis for the efficacy of methylselenocysteine (MSC) in increasing the therapeutic index of irinotecan against human tumor xenografts. Nude mice bearing human head and neck squamous cells carcinoma xenografts (FaDu and A253) were treated orally with different doses of MSC and irinotecan. Plasma, tumor and normal tissue samples were collected at different times after MSC treatments and were analyzed for selenium (Se) concentration using electrothermal atomic absorption spectrophotometry. MSC is highly effective in modulating the therapeutic index of irinotecan. Enhanced irinotecan efficacy was greater in FaDu tumors (100% CR) than in A253 tumors (60% CR), and depended on MSC dose with a minimum effective dose of 0.01mg/d×28. The highest plasma Se concentration was achieved 1h after a single dose and 28 d after daily treatments of MSC. The ability of FaDu tumors to retain Se was significantly better than A253 tumors, and the highest Se concentration in normal tissue was achieved in the liver. Peak plasma and tissue Se concentrations were functions of the dose and duration of MSC treatment. The MSC-dependent increase in Se level in normal tissues may contribute to the protective effect against irinotecan toxicity observed in those tissues. Intratumoral total Se concentration was not found to be predictive of the combination therapy response rates. There is a critical need to develop a method to measure the active metabolite of MSC, rather than total Se.

Keywords: Abbreviations; MSC; methylselenocysteine; Se; selenium; MTD; maximum tolerated dose; CR; cure rates; i.v.; intravenously; s.c.; subcutaneously; QC; quality control; AUC; area under the concentration time curveMethylselenocysteine; Selenium; FaDu; A253; Irinotecan

Cancer chemopreventive properties of orally bioavailable flavonoids—Methylated versus unmethylated flavones by Thomas Walle; Nga Ta; Toshihiko Kawamori; Xia Wen; Petra A. Tsuji; U. Kristina Walle (pp. 1288-1296).

Poor oral bioavailability has been a major limitation for the successful use of dietary flavonoids as cancer chemopreventive agents. In this study, we examined fully methylated flavones as promising improved agents. In the human oral SCC-9 cancer cells, 5,7-dimethoxyflavone and 5,7,4′-trimethoxyflavone were both 10 times more potent inhibitors of cell proliferation (IC₅₀ values 5–8μM) than the corresponding unmethylated analogs chrysin and apigenin. Flow cytometry indicated that both methylated flavones arrested the SCC-9 cells in the G1 phase with a concomitant decrease in the S phase, dramatically different from the unmethylated analogs, which promoted G2/M phase arrest. Both methylated compounds inhibited the proliferation of two other cancer cell lines with very little effect on two immortalized normal cell lines. Examination of additional flavone structures indicated that methylated flavones in general have antiproliferative properties. Finally, we demonstrated that 5,7-dimethoxyflavone, in contrast to its unmethylated analog chrysin, was well absorbed and had high oral bioavailability as well as tissue accumulation in vivo in the rat. Thus, fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents, in particular in oral cancer.

Keywords: Abbreviations; SCC; squamous cell carcinoma; DMF; dimethoxyflavone; TMF; trimethoxyflavone; DHF; dihydroxyflavone; MF; methoxyflavone; HF; hydroxyflavone; PMF; pentamethoxyflavone; BrdU; bromodeoxyuridine; FBS; fetal bovine serum; DMSO; dimethyl sulfoxide; PBS; phosphate-buffered salineFlavonoids; Methylated flavones; Cancer prevention; Proliferation; Bioavailability

Histone deacetylase inhibitor Trichostatin A induces global and gene-specific DNA demethylation in human cancer cell lines by Jing-Ni Ou; Jérôme Torrisani; Alexander Unterberger; Nadine Provençal; Keisuke Shikimi; Mohsen Karimi; Tomas J. Ekström; Moshe Szyf (pp. 1297-1307).

DNA methylation and chromatin structure are two modes of epigenetic control of genome function. Although it is now well established that chromatin silencing could lead to DNA methylation, the relation between chromatin activation and DNA demethylation is unclear. It was generally believed that expression of methylated genes could only be restored by demethylating agents, such as 5-aza-deoxycytidine (5-azaCdR), and that inhibition of histone deacetylation by Trichostatin A (TSA) only activates transcription of unmethylated genes. In this report, we show that increase of histone acetylation by TSA was associated with a significant decrease in global methylation. This global demethylation occurs even when DNA replication is blocked by hydroxyurea, supporting a replication-independent-mechanism of demethylation. TSA also induces histone acetylation, demethylation and expression of the methylated E-CADHERIN and RARβ2 genes. However, the genome-wide demethylation induced by TSA does not affect all methylated tumor suppressor genes equally suggesting that induction of acetylation and demethylation by TSA shows some gene selectivity. Taken together, our data provide evidence for a reversible crosstalk between histone acetylation and DNA demethylation, which has significant implications on the use of HDAC inhibitors as therapeutic agents.

Keywords: Abbreviations; TSA; Trichostatin A; 5-azaCdR; 5-aza-deoxycytidine; HDACi; histone deacetylase inhibitor; HAT; histone acetyltransferase; DNMT; DNA methyltransferaseHistone deacetylase inhibitor; Tumor suppressor gene; DNA methylation; Histone acetylation; Hypomethylation; Hydroxyurea

Sorafenib alone or as combination therapy for growth control of cholangiocarcinoma by Alexander Huether; Michael Höpfner; Viola Baradari; Detlef Schuppan; Hans Scherübl (pp. 1308-1317).

Treatment options of advanced cholangiocarcinoma (CC) are unsatisfactory and new therapeutic approaches are mandatory. Dysregulations of the mitogen-activated kinase (MAPK) pathway associated with proliferative advantages of tumors are commonly observed in CCs. The novel multi-kinase inhibitor sorafenib potently suppresses the growth of various cancers by inhibiting kinases of wild-type B-Raf, mutant^V559EB-Raf and C-Raf but its effects on CC remains to be explored. We therefore studied the antineoplastic potency of sorafenib in human CC cells alone and in combination with conventional cytostatics or IGF-1R inhibition.Sorafenib treatment dose-dependently blocked growth-factor-induced activation of the MAPKP and inhibited the proliferation of EGI-1 and TFK-1 CC cells in a time- and dose-dependent manner. At least two mechanisms accounted for the effects observed: arrest at the G₁/G₀-transition of the cell cycle and induction of apoptosis. The cell cycle arrest was associated with upregulation of the cyclin-dependent kinase inhibitor p27^Kip1 and downregulation of cyclin D1. Combining sorafenib with doxorubicin or IGF-1R-inhibition resulted in (over)additive antiproliferative effects whereas co-application of sorafenib and the antimetabolites 5-FU or gemcitabine diminished the antineoplastic effects of the cytostatics.Our study demonstrates that the growth of human CC cells can be potently suppressed by sorafenib alone or in certain combination therapies and may provide a promising rationale for future in vivo evaluations and clinical trials.

Keywords: Abbreviations; AG1024; 3-bromo-5-; t; -butyl-4-hydroxy-benzylidenemalonitrile; CC; cholangiocarcinoma; EGF; epidermal growth factor; ERK1/2; extracellular regulated kinase 1/2; 5-FU; 5-flourouracil; IGF; insulin-like growth factor; IGF-1R; insulin-like growth factor 1 receptor; MAPK; mitogen activated kinase; MAPKP; mitogen activated kinase pathway; sorafenib; N; -(3-trifluoromethyl-4-chlorophenyl)-; N; ′-(4-(2-methylcarbamoyl-pyridin-4-yl)oxyphenyl)ureaNexavar™; ERK1/2; Insulin-like growth factor receptor 1; Combination treatment; EGI-1; TFK-1

Celecoxib inhibits the expression of survivin via the suppression of promoter activity in human colon cancer cells by Naoko Sakoguchi-Okada; Fumi Takahashi-Yanaga; Kazuhiro Fukada; Fumie Shiraishi; Yoji Taba; Yoshikazu Miwa; Sachio Morimoto; Mitsuo Iida; Toshiyuki Sasaguri (pp. 1318-1329).

We investigated the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on human colon cancer cell lines to clarify the mechanisms underlying the chemopreventive effect of NSAIDs. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, induced apoptosis and strongly reduced the expression of an anti-apoptotic protein, survivin, in both protein and mRNA levels in HCT-116 cells. Subsequently, we conducted luciferase reporter assay using a reporter gene driven by the human survivin promoter. A series of analyses using luciferase reporter constructs containing fragments of the survivin promoter and electrophoretic mobility shift assay indicated that the −75/−66bp region relative to the initiating codon was involved in celecoxib action to suppress survivin promoter activity. Celecoxib also suppressed the activity of TOPflash, T-cell factor reporter plasmid, and the reporter gene driven by the human cyclin D1 promoter, suggesting that this compound inhibited the expression of Wnt/β-catenin signaling target genes. Further, we found that other NSAIDs including indomethacin, resveratrol, and SC-560 induced apoptosis and suppressed the expression of survivin and the Wnt/β-catenin signaling pathway in HCT-116 cells, indicating that these effects were likely to be common among NSAIDs. Moreover, NSAIDs (celecoxib, SC-560 and indomethacin) also suppressed the expression of cyclin D1 and survivin on other colon cancer cell lines (DLD-1 and SW-620). Our results suggested that NSAIDs could inhibit proliferation and induce apoptosis in colon cancer cells by inhibition of survivin expression and the Wnt/β-catenin signaling pathway.

Keywords: Abbreviations; NSAIDs; nonsteroidal anti-inflammatory drugs; COX-2; cyclooxygenase-2; FBS; fetal bovine serum; TCF; T-cell factorCelecoxib; Survivin; Promoter activity; Apoptosis; Colon cancer cell; Nonsteroidal anti-inflammatory drugs (NSAIDs)

Tamoxifen and estrogen attenuate enhanced vascular reactivity induced by estrogen deficiency in rat carotid arteries by Suk Ying Tsang; Xiaoqiang Yao; Hoi Yun Chan; Franky Leung Chan; Cecilia Sze-Lee Leung; Lai Ming Yung; Chak Leung Au; Zhen-Yu Chen; Ismail Laher; Yu Huang (pp. 1330-1339).

Recent clinical trials showed that estrogen usage in postmenopausal women did not affect coronary heart disease incidence, in contrast to several laboratory studies showing that estrogen decreased vascular reactivity. We speculated that, in some arteries, estrogen deficiency enhances endothelial function to compensate for the increased vascular smooth muscle reactivity. In this study, we examined the role of endothelium-derived vasoactive factors and the influence of in vivo estrogen and/or tamoxifen treatment on vascular reactivity of estrogen-deficient rats. Common carotid arteries were isolated from sham-operated (control), ovariectomized (Ovx), estrogen- or tamoxifen-treated Ovx rats, and Ovx rats co-treated with estrogen and tamoxifen. U46619 or phenylephrine induced similar contractions in endothelium-intact rings from all groups. Interestingly, removal of endothelium unmasked enhanced contractions in Ovx rats, which was prevented by estrogen, tamoxifen, or estrogen+tamoxifen treatment. Contractions to high K⁺ were higher in both endothelium-intact and endothelium-denuded arteries from Ovx rats. Estrogen or tamoxifen treatment normalized high K⁺-induced contraction. A gap junction blocker, 18α-glycyrrhetinic acid, revealed enhanced contractions to U46619 in the absence or presence ofl-NNA. Western blotting showed enhanced expressions of gap junctional connexin 43 in Ovx group. This study suggests that ovariectomy increases functional expression of gap junction-mediated endothelium-derived hyperpolarizing factor. Also, vascular effects of ovariectomy can be reversed by estrogen, tamoxifen or estrogen+tamoxifen treatment, suggesting that tamoxifen confers estrogenic effects in the vascular system.

Keywords: Abbreviations; CCA; common carotid artery; CTX; charybdotoxin; EDHF; endothelium-derived hyperpolarizing factor; HRT; hormone replacement therapy; IBX; iberiotoxin; l; -NNA; N; ^G; -nitro-; l; -arginine; NO; nitric oxide; NOS; nitric oxide synthase; Ovx; ovariectomy or ovariectomized; PGI; ₂; prostacyclin; SERM; selective estrogen receptor modulator; TEA; ⁺; tetraethylammonium; U46619; 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F; _2αChronic estrogen; Chronic tamoxifen; Common carotid arteries; Vascular reactivity; Endothelium-derived hyperpolarizing factor

Inhibition of atherosclerosis by the serine palmitoyl transferase inhibitor myriocin is associated with reduced plasma glycosphingolipid concentration by Elias N. Glaros; Woojin S. Kim; Benjamin J. Wu; Cacang Suarna; Carmel M. Quinn; Kerry-Anne Rye; Roland Stocker; Wendy Jessup; Brett Garner (pp. 1340-1346).

Glycosphingolipids (GSL) have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels in the absence of changes in cholesterol or triglyceride (TG) concentration and this leads to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE^−/−) mice. The possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma GSL concentration has not been examined and was the primary aim of this investigation. We show that intraperitoneal delivery of the SPT inhibitor myriocin for 9 weeks inhibits atherosclerosis in apoE^−/− mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentration of 42% and 25%, as assessed by enzymatic and HPLC methods, respectively. Moreover, SM and GSL concentrations were significantly correlated, indicating that SPT inhibition suppresses the synthesis of both these sphingolipids concomitantly. The inhibition of atherosclerosis induced by myriocin was not associated with changes in plasma cholesterol or TG concentrations or lipoprotein profiles as determined by FPLC. These data indicate that therapeutic reduction of plasma SM and/or GSL concentrations may offer a novel treatment for atherosclerosis.

Keywords: Atherosclerosis; Sphingolipid inhibition; Glycosphingolipids; Myriocin/ISP-1; Apolipoprotein-E knockout mouse

The activation by estrogen receptor agonists of the BK_Ca-channel in human cardiac fibroblasts by Ya-Jean Wang; Ming-Wei Lin; Sheng-Nan Wu; Ruey J. Sung (pp. 1347-1357).

The agonists selective for estrogen receptor (ER)-α (4,4′,4′′-(4-propyl-[¹H]-pyrazole-1,3,5-triyl) tris-phenol, PPT) and ER-β (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN) are known to stimulate ER-α and ER-β receptors, respectively. It remains unknown whether these two agents regulate the activity of ion channels via a direct stimulation. In this study, we tested the hypothesis that DPN or PPT stimulates the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human cardiac fibroblasts (HCFs). In whole-cell configuration, depolarizing pulses evoked K⁺ outward currents in an outward rectification in HCFs, the amplitude of which was increased in the presence of DPN or PPT. In inside-out patches, the activity of BK_Ca-channel with a conductance of 167±8pS was observed in these cells. PPT or DPN applied to the intracellular face of the membrane enhanced the activity of BK_Ca channels with no change in single-channel conductance. DPN and PPT increased BK_Ca-channel activity with an EC₅₀ value of 2.3 and 2.6μM, respectively. The mean closed time of these channels during the exposure to these compounds was reduced with no change in the gating charge of the channels. Intracellular Ca²⁺ was not altered by these two compounds. RT-PCR analysis revealed that no change in the transcriptional level of the BK_Ca-channel α-subunit was observed in chronic treatment with these two compounds. PPT- and DPN-stimulated increase in BK_Ca channels reveal novel pharmacological properties attributable to the activity of these channels, and their increase in BK_Ca channels activity in HCFs may contribute to cell function.

Keywords: Abbreviations; BK; _Ca; channel; large-conductance Ca; ²⁺; -activated K; ⁺; channel; [Ca; ²⁺; ]; _i; intracellular Ca; ²⁺; DMSO; dimethyl sulfoxide; DPN; 2,3-bis(4-hydroxyphenyl)-propionitrile; PPT; 4,4′,4′′-(4-propyl-[; ¹; H]-pyrazole-1,3,5-triyl) tris-phenol; ER; estrogen receptor; GADPH; glyceraldehydes-3-phosphate dehydrogenase; HCF; human cardiac fibroblast; I; _K; K; ⁺; outward current; I; -; V; current-voltage; RT-PCR; reverse transcription-polymerase chain reactionK; ⁺; outward current; Large-conductance Ca; ²⁺; -activated K; ⁺; channel; Estrogen receptor agonist; Human cardiac fibroblast

Nrf2 is involved in the effect of tanshinone IIA on intracellular redox status in human aortic smooth muscle cells by Hong-Sheng Zhang; Sheng-Qi Wang (pp. 1358-1366).

Tanshinone IIA is the major antioxidant component in the traditional Chinese medicine Salvia miltiorrhiza. Transcription factor nuclear-factor-E2-related factor (Nrf2) regulates a battery of antioxidant response element (ARE)-regulated genes. The aim of this study was to determine the effect of tanshinone IIA on Nrf2 activation and intracellular redox status in human aortic smooth muscle cells. Tanshinone IIA potentiated tumor necrosis factor α (TNF-α)-mediated nuclear accumulation of Nrf2 and expression of ARE-related genes, while it reversed TNF-α-induced down-regulation of intracellular glutathione (GSH), NADPH and glucose 6-phosphate dehydrogenase (G6PDH) levels. Specific silence of Nrf2 by siRNA down-regulated tanshinone IIA-induced Nrf2 activation and increased of intracellular GSH, NADPH and G6PDH levels. Tanshinone IIA-induced Nrf2 activation was association with activation of ERK and PKB, which was prevented by treatment with PD098059 or wortmannin. Tanshinone IIA attenuated TNF-α, angiotensin II, H₂O₂-mediated reactive oxygen species (ROS) production. These results demonstrated that tanshinone IIA-induced Nrf2 activation is the major regulatory pathway of cytoprotective gene expression against oxidative stress via ERK and PKB signaling pathways.

Keywords: Tanshinone IIA; Nrf2; Oxidative stress; Smooth muscle cells

Improved endothelial function and reduced platelet activation by chronic HMG-CoA-reductase inhibition with rosuvastatin in rats with streptozotocin-induced diabetes mellitus by Andreas Schäfer; Daniela Fraccarollo; Christian Vogt; Ulrike Flierl; Melinda Hemberger; Piet Tas; Georg Ertl; Johann Bauersachs (pp. 1367-1375).

Diabetes is associated with endothelial dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. We investigated whether the hydroxy-3-methyl-glutaryl CoA reductase inhibitor rosuvastatin improves endothelial function and reduces platelet activation in diabetic rats. Therefore, male Wistar rats were injected with streptozotocin (STZ, 50mg/kg i.v.) to induce insulin-deficient diabetes. Treatment with rosuvastatin (20mg/[kgday]) or vehicle was initiated 2 weeks after injection of STZ and continued for 2 weeks. Thereafter, platelet activation was assessed in fresh whole blood and vascular function was characterized in isolated aortic segments in organ bath chambers. Endothelium-dependent relaxation induced by acetylcholine was significantly attenuated in diabetic rats and improved by treatment with rosuvastatin (maximum relaxation, % of precontraction—control: 99.8±0.2, STZ-vehicle: 80.7±2.9, STZ-rosuvastatin: 98.9±0.7; p<0.01). Similarly, treatment with rosuvastatin significantly reduced fibrinogen-binding to activated GPIIb/IIIa (mean fluorescence—control: 161.0±6.9, STZ-vehicle: 207.8±15.9, rosuvastatin: 173.6±5.3; p<0.05) and P-Selectin surface expression on platelets (mean fluorescence—control: 76.5±7.3, STZ-vehicle: 92.1±5.5, rosuvastatin: 75.2±6.5; p<0.05), while both markers of platelet activation were increased in diabetic rats. Therefore, rosuvastatin treatment normalizes endothelial function and reduces platelet activation in diabetic rats. These effects may contribute to the reduction of cardiovascular events by statins in diabetic patients.

Keywords: Endothelial dysfunction; Nitric oxide; Diabetes; Platelets; Statins

Selective action of the iminosugar isofagomine, a pharmacological chaperone for mutant forms of acid-β-glucosidase by Richard Steet; Stephen Chung; Wang-Sik Lee; Corey W. Pine; Hung Do; Stuart Kornfeld (pp. 1376-1383).

Gaucher disease is a lysosomal glycolipid storage disorder characterized by defects in acid-β-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. We recently demonstrated that isofagomine (IFG), an iminosugar that binds to the active site of GlcCerase, enhances the folding, transport and activity of the N370S mutant form of GlcCerase. In this study we compared the effects of IFG on a number of other glucosidases and glucosyltransferases. We report that IFG has little or no inhibitory activity towards intestinal disaccharidase enzymes, ER α-glucosidase II or glucosylceramide synthase at concentrations previously shown to enhance N370S GlcCerase folding and trafficking in Gaucher fibroblasts. Furthermore, treatment of wild type fibroblasts with high doses of IFG did not alter the processing of newly synthesized N-linked oligosaccharides. These findings support further evaluation of IFG as a potential therapeutic agent in the treatment of some forms of Gaucher disease.

Keywords: Abbreviations; GlcCerase; acid-β-glucosidase; IFG; isofagomine; NB-DNJ; N; -butyldeoxynojirimycinIsofagomine; N; -Butyldeoxynojirimycin; Gaucher; Acid-β-glucosidase; Disaccharidase; α-Glucosidase II

In vitro characterization of the effects of endomorphin 1 and 2, endogenous ligands for μ-opioid receptors, on mouse colonic motility by Ye Yu; Yun Cui; Xiang Wang; Lu-hao Lai; Chang-Lin Wang; Ying-zhe Fan; Jing Liu; Rui Wang (pp. 1384-1393).

The effects of endomorphin 1 (EM1) and 2 (EM2) in colonic motility remain unknown. We investigated the effects and mechanisms of these endomorphins (EMs) on the colonic motility in vitro by applying various neural blocking agents and various opioid receptor antagonists. EMs (10⁻⁹ to 10⁻⁶M) displayed significant stimulatory effects on the basal tonus or spontaneous activity of mouse colon but not of stomach and small intestine. It is noteworthy that the contractile actions of EMs varied slightly among different regions of colonic longitudinal muscle layers, whereas the contractile responses induced by EMs were significantly different among different regions of circular muscle layers. EMs-induced longitudinal or circular muscle contractions were not significantly affected by atropine, N^G-nitro-l-arginine methyl ester, phentolamine, propranolol and methysergide. Tetrodotoxin, indomethacin and naloxone completely abolished the EMs-induced colonic contractions. Surprisingly, EMs (10⁻⁷M)-induced longitudinal muscle contractions were significantly attenuated by nor-binaltorphimine (3×10⁻⁶M). By contrast, pretreatment with naltrindole (10⁻⁶M) did not significantly affect EMs-induced longitudinal or circular muscle contractions. Interestingly, the circular muscle contractions in response to EM2 (10⁻⁷M) were not fully blocked by β-funaltrexamine (6×10⁻⁶M). Naloxonazine (10⁻⁶M) almost fully antagonized the EMs-induced longitudinal or circular muscle contractions, and these effects could be only partially reversed by extensive washing. All the results indicated that the mechanisms and sites of actions of EMs were region-specific. Furthermore, these findings showed that the activation of multiple subtypes of opioid receptors, possibly including μ₁ (naloxonazine-sensitive), μ₂ and even other forms of μORs (β-FNA-insensitive), was required for EMs-induced mouse colonic motility.

Keywords: Abbreviations; DAMGO; [; d; -Ala; ²; N-Me-Phe; ⁴; Gly-ol; ⁵; ]-enkephalin; DMSO; dimethylsulfoxide; EM1; endomorphin 1; EM2; endomorphin 2; EMs; endomorphins; β-FNA; β-funaltrexamine; GI; gastrointestinal; l; -NAME; N; ^G; -nitro-; l; -arginine methyl ester; nor-BNI; nor-binaltorphimine; NTI; naltrindole; ORs; opioid receptorsEndomorphin 1; Endomorphin 2; Colonic motility; Opioid receptors antagonists; Neural blocking agents

Novel bile acid derivatives (BANBs) with cytostatic activity obtained by conjugation of their side chain with nitrogenated bases by Marta Vallejo; Maria A. Castro; Manuel Medarde; Rocio I.R. Macias; Marta R. Romero; Mohamad Y. El-Mir; Maria J. Monte; Oscar Briz; Maria A. Serrano; Jose J.G. Marin (pp. 1394-1404).

Drug targeting may contribute to overcoming resistance to chemotherapy and to reducing side effects. Here, by conjugating a nitrogenated base (NB) to the side chain of a bile acid (BA) moiety, we have synthesized and evaluated six novel compounds, designated BANB-1 to -6, with potential cytostatic activity and vectoriality toward enterohepatic tumors. These compounds were purified by liquid chromatography and their purity was checked by TLC and HPLC before being chemically characterized using IR,¹H/¹³C NMR and FAB-MS. Using several cell lines – HepG2 (human hepatoblastoma), LS 174T and Caco-2 (human colon adenocarcinoma), Hepa 1-6 (mouse hepatoma), McA-RH7777 (rat hepatoma), CCRF S-180 II (mouse sarcoma) and CHO (Chinese hamster ovary) – their effect on cell viability was measured with the formazan test after drug exposure for 6h (cytotoxic effect) or 72h (cytostatic effect). A weak cytostatic effect of BANB-1, BANB-2 and BANB-3 was detected even in CHO cells stably transfected with rat bile acid transporters (Ntcp and Oatp1/1a1). In contrast, BANB-4, BANB-5 and BANB-6, similarly to cisplatin, showed strong cytostatic effects, together with mild non-specific toxicity. BANB-6 was effective even against Hepa 1-6/R cells, which were partly resistant to cisplatin. Treatment with BANB-6, but not cisplatin, was able to prolong the life span of Nude mice bearing tumors formed by Hepa 1-6/R cells orthotopically implanted in the liver. In conclusion, our results support the hypothesis that cytostatic bile acid derivatives such as BANB-6 may offer a useful pharmacological strategy for the treatment of tumors of the enterohepatic circuit.

Keywords: Abbreviations; ASBT; apical sodium-dependent bile acid transporter; BA; bile acid; BANB; bile acid-nitrogenated base conjugate; CA; cholic acid; FAB; fast atom bombardment; GCA; glycocholic acid; HRMS; high-resolution mass spectrometry; MDR; multidrug resistance protein; MRP; multidrug resistance-associated protein; NB; nitrogenated base; NMR; nuclear magnetic resonance; NTCP or Ntcp; human or rat sodium-taurocholate cotransporting polypeptide; OATP or Oatp; human or rat organic anion-transporting polypeptide; OCT; organic cation transporter; OST; organic solute transporter; UDCA; ursodeoxycholic acidCancer; Chemotherapy; Colon; Gut; Intestine; Liver; Resistance; Tumor

An antioxidant effect by acyclic retinoid suppresses liver tumor in mice by Tomohiko Sakabe; Hiroyuki Tsuchiya; Michiko Endo; Akiko Tomita; Kyoko Ishii; Kazue Gonda; Rie Murai; Kazuko Takubo; Yoshiko Hoshikawa; Akihiro Kurimasa; Naoto Ishibashi; Shingo Yanagida; Goshi Shiota (pp. 1405-1411).

The mechanisms of prevention of the development of liver cancer by NIK-333, an acyclic retinoid (ACR), were investigated. The transgenic mice expressing the dominant negative form of retinoic acid receptor α (RARE mice), that produce reactive oxygen species and lead to development of liver tumor were used. The effect of NIK-333 on hepatocarcinogenesis in RARE mice was studied. The RARE mice were examined after feeding 0.03% and 0.06% NIK-333 diets at 12 months of age. In the mice fed 0.06% NIK-333 diet, tumor incidence was greatly suppressed, compared to that of wild type mice (0/9 versus 5/9, P<0.05), but not in the mice fed 0.03% NIK-333 diet. In addition, expression of cytochrome p450 4a14 and acyl-CoA oxidase was normalized, and the percentages of positive cells for 8-hydroxy-2′-deoxyguanosine, 4-hydroxy-2-nonenal and proliferating cell nuclear antigen were decreased. Furthermore, expression of β-catenin and cyclin D1 was also depressed. These data suggest that NIK-333 suppressed liver tumor in association with repression of oxidative stress.

Keywords: Abbreviations; ACR; acyclic retinoid; LCAD; long-acyl-CoA dehydrogenase; VLCAD; very long-acyl-CoA dehydrogenase; AOX; acly-CoA oxidase; CYP4a14; cytochrome p4504a14; 8-OHdG; 8-hydroxy-2′-deoxyguanosine; 4-HNE; 4-hydroxy-2-nonenal; PCNA; proliferating cell nuclear antigen; GAPDH; glyceraldehydes-3-phosphate dehydrogenaseAcyclic retinoid; Liver tumor; Antioxidative effect; Chemoprevention; Lipid metabolism; Oxidative stress

Rosmarinic acid inhibits indoleamine 2,3-dioxygenase expression in murine dendritic cells by Hwa Jung Lee; Young-Il Jeong; Tae-Hyung Lee; In Duk Jung; Jun Sik Lee; Chang-Min Lee; Jong-Il Kim; Hwan Joo; Jae-Dong Lee; Yeong-Min Park (pp. 1412-1421).

Indoleamine 2,3-dioxygenase (IDO), a key enzyme that catalyses the initial and rate-limiting step in the degradation of the tryptophan, is simultaneously expressed in murine dendritic cells and macrophages stimulated with interferon-γ (IFN-γ). In the present study, we investigated whether rosmarinic acid (RA), which is suggested to exhibit anti-oxidant and anti-cyclooxygenase properties, could suppress the functional expression of IDO in murine bone marrow-derived dendritic cells (BMDCs) stimulated with IFN-γ. Treatment with RA reduced intracellular expression of IDO both in IFN-γ-activated BMDCs in vitro and in CD11c⁺CD8α⁺ DCs in vivo tumor-bearing mice model. Consequently, we obtained evidence that RA suppresses the functional activity of IDO and blocks the IDO-dependent T cell suppression. In IFN-γ-mediated induction of IDO transcription, activation of the signal transducer and activator of transcription 1 (STAT1) is important to be express IDO in IFN-γ-stimulated BMDCs. In this study, we demonstrated that the RA could also suppress IFN-γ-induced STAT1 activation. These novel findings provide a new insight into that RA as a pharmacological and transcriptional inhibitor of IDO is worthy of clinical application as well as further investigation for IDO regulation.

Keywords: Indoleamine 2,3-dioxygenase; Dendritic cells; Rosmarinic acid; IFN-γ; Signal transducer and activator of transcription 1; T cell

Biomarker discovery for inflammatory bowel disease, using proteomic serum profiling by Marie-Alice Meuwis; Marianne Fillet; Pierre Geurts; Dominique de Seny; Laurence Lutteri; Jean-Paul Chapelle; Vincent Bours; Louis Wehenkel; Jacques Belaiche; Michel Malaise; Edouard Louis; Marie-Paule Merville (pp. 1422-1433).

Crohn's disease and ulcerative colitis known as inflammatory bowel diseases (IBD) are chronic immuno-inflammatory pathologies of the gastrointestinal tract. These diseases are multifactorial, polygenic and of unknown etiology. Clinical presentation is non-specific and diagnosis is based on clinical, endoscopic, radiological and histological criteria. Novel markers are needed to improve early diagnosis and classification of these pathologies. We performed a study with 120 serum samples collected from patients classified in 4 groups (30 Crohn, 30 ulcerative colitis, 30 inflammatory controls and 30 healthy controls) according to accredited criteria. We compared protein sera profiles obtained with a Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometer (SELDI-TOF-MS). Data analysis with univariate process and a multivariate statistical method based on multiple decision trees algorithms allowed us to select some potential biomarkers. Four of them were identified by mass spectrometry and antibody based methods. Multivariate analysis generated models that could classify samples with good sensitivity and specificity (minimum 80%) discriminating groups of patients. This analysis was used as a tool to classify peaks according to differences in level on spectra through the four categories of patients. Four biomarkers showing important diagnostic value were purified, identified (PF4, MRP8, FIBA and Hpα2) and two of these: PF4 and Hpα2 were detected in sera by classical methods. SELDI-TOF-MS technology and use of the multiple decision trees method led to protein biomarker patterns analysis and allowed the selection of potential individual biomarkers. Their downstream identification may reveal to be helpful for IBD classification and etiology understanding.

Keywords: Proteomics; IBD; Biomarkers; SELDI-TOF-MS; Inflammation; Serum profiling

Suppression of NF-κB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes: Implications for the treatment of osteoarthritis by Mehdi Shakibaei; Thilo John; Gundula Schulze-Tanzil; Ingo Lehmann; Ali Mobasheri (pp. 1434-1445).

Pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) play a key role in the pathogenesis of osteoarthritis (OA). Anti-inflammatory agents capable of suppressing the production and catabolic actions of these cytokines may have therapeutic potential in the treatment of OA and a range of other osteoarticular disorders. The purpose of this study was to examine the effects of curcumin (diferuloylmethane), a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1β and TNF-α signalling pathways in human articular chondrocytes maintained in vitro. The effects of curcumin were studied in cultures of human articular chondrocytes treated with IL-1β and TNF-α for up to 72h. Expression of collagen type II, integrin β1, cyclo-oxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) was monitored by western blotting. The effects of curcumin on the expression, phosphorylation and nuclear translocation of protein components of the NF-κB system were studied by western blotting and immunofluorescence, respectively. Treatment of chondrocytes with curcumin suppressed IL-1β-induced NF-κB activation via inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation and p65 nuclear translocation. Curcumin inhibited the IL-1β-induced stimulation of up-stream protein kinase B Akt. These events correlated with down-regulation of NF-κB targets including COX-2 and MMP-9. Similar results were obtained in chondrocytes stimulated with TNF-α. Curcumin also reversed the IL-1β-induced down-regulation of collagen type II and β1-integrin receptor expression. These results indicate that curcumin has nutritional potential as a naturally occurring anti-inflammatory agent for treating OA through suppression of NF-κB mediated IL-1β/TNF-α catabolic signalling pathways in chondrocytes.

Keywords: Chondrocyte; IL-1β; TNF-α; NF-κB; IκBα; Akt; Curcumin; Anti-inflammatory; Osteoarthritis (OA)

Mechanism of dopamine mediated inhibition of neuropeptide Y release from pheochromocytoma cells (PC12 cells) by Guihua Cao; Alice Gardner; Thomas C. Westfall (pp. 1446-1454).

In rat pheochromocytoma (PC12) cells the dopamine D₂ receptor agonists apomorphine (APO) and n-propylnorapomorphine (NPA) produced a concentration dependent inhibition of K⁺-evoked neuropeptide Y release (NPY-ir). The effect of APO was blocked by the dopamine D₂-receptor antagonist, eticlopride, but not the D₁/D₃ or the D₄/D₂ antagonists, SCH23390 or clozapine, respectively. The D₁/D₅ receptor agonist, SKF38393 or the D₃ agonists PD128907 and 7-OH DPAT had no effect. Selective N and L-type voltage gated Ca²⁺ channel blockers, ω-conotoxin GVIa (Ctx-GVIa) and nifedipine, respectively, produced a concentration dependent inhibition of NPY-ir release but were not additive with APO. The Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) II inhibitor KN-62 produced a concentration-dependent inhibition of NPY-ir release but the combination of KN-62 and APO produced no further inhibition. PMA-mediated protein kinase C stimulation significantly increased both basal and K⁺-evoked release of NPY-ir, and in the presence of PMA APO had no inhibitory effect. The PKC antagonist, chelerythrine, inhibited K⁺-evoked NPY-ir release but was not additive with APO. Neither forskolin-mediated adenylate cyclase activation and the active cAMP analog Sp-cAMPS, nor the adenylate cyclase inhibitor SQ 22536, and the competitive inhibitor of cAMP-dependent protein kinases Rp-cAMPS, had any significant effect on K⁺-evoked NPY-ir release. This suggests the inhibitory effect of APO on K⁺-evoked release of NPY-ir from PC12 cells is most likely mediated through activation of dopamine D₂ receptors leading to direct inhibition of N and L-type voltage gated Ca²⁺ channels, or indirect inhibition of PKC, both of which would reduce [Ca²⁺]_i and inactivate CaM kinase.

Keywords: Abbreviations; NPY-ir; neuropeptide Y-immunoreactivity; Ctx-GVIa; ω-conotoxin GVIaDopamine agonists; Neuropeptide Y; Dopamine receptors; Apomorphine; PC12 cells; Radioimmunoassay

Pharmacokinetics of a hepatic stellate cell-targeted doxorubicin construct in bile duct-ligated rats by Rick Greupink; Catharina Reker-Smit; Johannes H. Proost; Anne-miek van Loenen Weemaes; Marjolijn de Hooge; Klaas Poelstra; Leonie Beljaars (pp. 1455-1462).

Inhibition of hepatic stellate cell (HSC) proliferation is a relevant strategy to inhibit liver fibrosis. Coupling of antiproliferative drugs to the HSC-selective drug carrier mannose-6-phosphate-modified human serum albumin (M6PHSA) may lead to cell-selective inhibition of HSC proliferation. We coupled the antiproliferative drug doxorubicin (DOX) to this drug carrier and investigated the pharmacokinetics of this construct in a rat model of liver fibrosis, as well as in cultured HSC.M6PHSA-DOX was cleared from the plasma in a biphasic manner. Upon i.v. injection of 4μgkg⁻¹ (tracer), 2 and 20mgkg⁻¹, the clearance in the distribution phase of drug disposition (CL_d) significantly decreased from 9.7±0.7 to 4.7±2.3 and 1.0±0.1mlkg⁻¹min⁻¹, respectively. This indicates that saturation of clearance mechanisms occurs in this phase of drug disposition, likely reflecting saturable receptor-mediated uptake in the target cells. Gamma-camera studies revealed that the majority of the conjugate accumulated in the liver within 5min, and immunohistochemical double-staining of liver sections demonstrated co-localization of the construct with HSC-markers. Simulation of the release of DOX from the carrier, after cellular uptake by HSC, showed that a gradual release of the drug takes place over a 9h period. Studies in cultured HSC illustrated that after 24h incubation with the conjugate, DOX was associated with the cell nucleus.The rapid distribution of M6PHSA-DOX from the blood to HSC, in combination with the expected gradual release of DOX within these cells, make this construct a promising tool for achieving sustained and selective inhibition of HSC proliferation.

Keywords: Liver fibrosis; Drug targeting; Hepatic stellate cell; Selective delivery; Cytostatic drugs; Mannose-6-phosphate/insulin-like growth factor II receptor

Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation by Benjamin C. Lewis; Peter I. Mackenzie; David J. Elliot; Brian Burchell; C. Ramana Bhasker; John O. Miners (pp. 1463-1473).

Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7–UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61–194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11α-hydroxyprogesterone. Derived apparent K_m values for testosterone and phenolphthalein glucuronidation by UGT2B7-15_(61–194)-7 were similar in magnitude to those determined for UGT2B15. Moreover, glucuronidation of the non-selective substrate 4-methylumbelliferone (4MU) by UGT2B7-15_(61–194)-7 and UGT2B15 followed Michaelis–Menten and weak substrate inhibition kinetics, respectively, whereas 4MU glucuronidation by UGT2B7 exhibited sigmoidal kinetics characteristic of autoactivation. Six UGT2B7-15-7 chimeras that incorporated smaller domains of UGT2B15 were subsequently generated. Of these, UGT2B7-15_(61–157)-7, UGT2B7-15_(91–157)-7 and UGT2B7-15_(61–91)-7 glucuronidated 4MU, but activity towards the other substrates investigated here was not detected. Like UGT2B7, the UGT2B7-15_(61–157)-7, UGT2B7-15_(91–157)-7 and UGT2B7-15_(61–91)-7 chimeras exhibited sigmoidal 4MU glucuronidation kinetics. The sigmoidal 4MU kinetic data were well modelled using both the Hill equation and the expression for a two-site model that assumes the simultaneous binding of two substrate molecules at equivalent sites. It may be concluded that residues 61–194 of UGT2B15 are responsible for substrate binding and for conferring the unique substrate selectivity of UGT2B15, while residues 158–194 of UGT2B7 appear to facilitate the binding of multiple 4MU molecules within the active site.

Keywords: UDP-glucuronosyltransferase; Glucuronidation; Structure–function; Autoactivation; UGT2B7; UGT2B15

Redox regulation of human estrogen sulfotransferase (hSULT1E1) by Smarajit Maiti; Jimei Zhang; Guangping Chen (pp. 1474-1481).

Sulfotransferases (SULTs) are enzymes that catalyze the sulfation of hydroxyl-containing compounds. Sulfation regulates hormone activities and detoxifies xenobiotics. Human estrogen sulfotransferase (hSULT1E1) catalyzes the sulfation of estrogens and regulates estrogen bioactivities. Oxidative regulation provides a biological mechanism for regulating enzyme activities in vivo. The oxidative regulation of human SULTs has not been reported. In this study, we used amino acid modification, manipulation of intracellular redox state, and site-directed mutagenesis to study the redox regulation of human SULTs and specifically the mechanism of hSULT1E1 inhibitory regulation by oxidized glutathione (GSSG). Of the four major human SULTs, hSULT1A1, hSULT1A3, and hSULT2A1 do not undergo redox regulation; hSULT1E1, on the other hand, can be redox regulated. GSSG inactivated hSULT1E1 activity in an efficient, time- and concentration-dependant manner. The co-enzyme adenosine 3′-phosphate 5′-phosphosulfate protected hSULT1E1 from GSSG-associated inactivation. A reduced glutathione (GSH) inducer ( N-acetyl cysteine) significantly increased while a GSH depletor (buthionine sulfoxamine) significantly decreased hSULT1E1 activity, but both failed to affect the amount of hSULT1E1 protein in human hepatocyte carcinoma Hep G2 cells. Crystal structure suggested that no Cys residues exist near the active sites of hSULT1A1, hSULT1A3, and hSULT2A1, but Cys residues do exist within the active site of hSULT1E1. Site-directed mutagenesis demonstrated that Cys83 is critical for the redox regulation of hSULT1E1. This first report on the redox regulation of human SULTs suggests that the redox regulation of hSULT1E1 may interrupt the regulation and function of estrogens under various physiological and pathological conditions.

Keywords: Abbreviations; SULT; sulfotransferase; SULT1E1; estrogen sulfotransferase; NAC; N; -acetyl cysteine; BSO; buthionine sulfoxamine; PAPS; adenosine 3′-phosphate 5′-phosphosulfate; E; ₂; 17β-estradiol; ER; estrogen receptor; ROS; reactive oxygen species; RNS; reactive nitrogen speciesSulfotransferase; SULT1E1; Estrogen sulfotransferase; Redox regulation; Estrogen

Pharmacokinetic significance of luminal multidrug and toxin extrusion 1 in chronic renal failure rats by Kumiko Nishihara; Satohiro Masuda; Lin Ji; Toshiya Katsura; Ken-Ichi Inui (pp. 1482-1490).

Functional and expressional depression of the rat organic cation transporter rOCT2 after 5/6 nephrectomy (Nx) is accompanied by the decreased plasma level of testosterone in the male rats. Though vectorial transport across the tubular epithelial cells is important in the secretion of cationic compounds, there has been no imformation about the luminal organic cation transporter in disease state. In the present study, the role of luminal multidrug and toxin extrusion 1 (rMATE1) was examined using female rats with or without Nx, avoiding the influence of testosterone. The tubular secretion of cimetidine was markedly decreased in female Nx rats as well as male rats. Unlike in the male rats, the plasma level of testosterone and the expression of basolateral rOCT2 were unchanged in the female rats after Nx. On the other hand, the expression of rMATE1 was markedly decreased in both male and female Nx rats, and the level of rMATE1, but not of rOCT2, correlated well with the tubular secretion of cimetidine in the female rats ( r=0.74). Immunohistochemical analysis revealed that rMATE1 and Na⁺/H⁺ exchanger (NHE) 3 were localized at the brush-border membrane of proximal tubules. The level of NHE3 was also markedly depressed in both male and female Nx rats, suggesting that the expression level on the luminal rMATE1 in combination with NHE3 was indicated to be a crucial factor for the tubular secretion of cimetidine.

Keywords: Transporter; MATE1; Na; ⁺; /H; ⁺; exchanger 3; 5/6 Nephrectomy; Tubular secretion; Cimetidine

Alterations in cytoskeletal protein expression by mycophenolic acid in human mesangial cells requires Rac inactivation by Magali Mondin; Violaine Moreau; Elisabeth Genot; Christian Combe; Jean Ripoche; Isabelle Dubus (pp. 1491-1498).

In response to glomerular injury, mesangial cells are activated into myofibroblasts, which contribute to the physiopathology of glomerulosclerosis. We have previously shown that chronic treatment of cultured human mesangial cells with mycophenolic acid (MPA), a specific inhibitor of guanosine nucleotide synthesis, prevents their activation and alters cytoskeleton protein expression and associated functions, such as contractility and migratory capacity. The aim of the present study was to explore the mechanisms underlying MPA-induced mesangial cytoskeleton alterations.We therein show that coincubation with guanosine (100μM) compensates for the effects of MPA on mesangial cell proliferation and migration, and prevents MPA-induced overexpression of alpha-smooth muscle actin (SMA) and basic calponin (b-calp), indicating that guanylates are involved in mesangial responses to MPA. MPA decreased the GTP-bound (active) form of both RhoA, Rac1 and Cdc42, and specifically altered the expression level of Rac1. Pharmacological inhibition of RhoA activity reduced expression of both SMA and calponin, whereas overexpression of a dominant-negative form of Rac1 increased SMA expression. Conversely, overexpression of constitutively active Rac1 resulted in SMA and b-calp down-regulation, and fully prevented their stimulation by MPA, indicating that Rac inactivation is responsible for MPA effects on mesangial cytoskeletal expression.These results show that in human mesangial cells, RhoA and Rac1 exert opposite effects on the expression of two major cytoskeletal proteins: SMA and basic calponin. Moreover, these data highlight for the first time an integrated mechanism whereby MPA regulates mesangial phenotype, which is mediated by loss of Rac activity.

Keywords: Mycophenolic acid; Cytoskeleton; Mesangial cell; Glomerulosclerosis; Transplantation; Rho GTPases

Effects of hydroxyl radical scavenging on cisplatin-induced p53 activation, tubular cell apoptosis and nephrotoxicity by Man Jiang; Qingqing Wei; Navjotsin Pabla; Guie Dong; Cong-Yi Wang; Tianxin Yang; Sylvia B. Smith; Zheng Dong (pp. 1499-1510).

Nephrotoxicity is a major side effect of cisplatin, a widely used cancer therapy drug. Recent work has suggested a role of p53 in renal cell injury by cisplatin. However, the mechanism of p53 activation by cisplatin is unclear. This study determined the possible involvement of oxidative stress in p53 activation under the pathological condition using in vitro and in vivo models. In cultured renal proximal tubular cells, cisplatin at 20μM induced an early p53 phosphorylation followed by protein accumulation. Cisplatin also induced reactive oxygen species (ROS), among which hydroxyl radicals showed a rapid and drastic accumulation. Dimethylthiourea (DMTU) and N-acetyl-cysteine (NAC) attenuated hydroxyl radical accumulation, and importantly, diminished p53 activation during cisplatin treatment. This was accompanied by the suppression of PUMA-α, a p53-regulated apoptotic gene. Concomitantly, mitochondrial cytochrome c release and apoptosis were ameliorated. Notably, DMTU and NAC, when added post-cisplatin treatment, were also inhibitory to p53 activation and apoptosis. In C57BL/6 mice, cisplatin at 30mg/kg induced p53 phosphorylation and protein accumulation, which was also abrogated by DMTU. DMTU also ameliorated tissue damage, tubular cell apoptosis and cisplatin-induced renal failure. Collectively, this study has suggested a role of oxidative stress, particularly hydroxyl radicals, in cisplatin-induced p53 activation, tubular cell apoptosis and nephrotoxicity.

Keywords: Cisplatin; Nephrotoxicity; Apoptosis; p53; Hydroxyl radical; Oxidative stress

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Biochemical Pharmacology (v.73, #9)