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Biochemical Pharmacology (v.73, #1)

Editorial Advisory Board (pp. iii).

PAX genes: Roles in development, pathophysiology, and cancer by Deborah Lang; Sara K. Powell; Rebecca S. Plummer; Kacey P. Young; Bruce A. Ruggeri (pp. 1-14).

PAX proteins function as transcription factors and play an essential role in organogenesis during embryonic development in regulating cell proliferation and self-renewal, resistance to apoptosis, migration of embryonic precursor cells, and the coordination of specific differentiation programs. Recent studies have also discovered a role for PAX proteins in specific stem cell or progenitor cell populations, including melanocytes, muscle, and B-cells. The normal functions of the PAX proteins, including apoptosis resistance and repression of terminal differentiation, may be subverted during the progression of a number of specific malignancies. This is supported by the fact that expression of PAX proteins is dysregulated in several different types of tumors, although the precise roles for PAX proteins in cancer are not clearly understood. An emerging hypothesis is that PAX proteins play an essential role in maintaining tissue specific stem cells by inhibiting terminal differentiation and apoptosis and that these functional characteristics may facilitate the development and progression of specific cancers. In this review, we provide a general background to the PAX protein family and focus on specific cells and tissues and the role PAX proteins play within these tissues in terms of development, mature tissue maintenance, and expression in tumors. Understanding the normal developmental pathways regulated by PAX proteins may shed light on potentially parallel pathways shared in tumors, and ultimately result in defining new molecular targets and signaling pathways for the development of novel anti-cancer therapies.

Keywords: PAX genes; Embryological development; Cancer progression

Blockade of AKT activation in prostate cancer cells with a small molecule inhibitor, 9-chloro-2-methylellipticinium acetate (CMEP) by Manchao Zhang; Xueliang Fang; Hongpeng Liu; Shaomeng Wang; Dajun Yang (pp. 15-24).

AKT inhibitors are potentially promising drug candidates for the treatment of cancer. The inhibitory effects of a potent and selective AKT/BKB small molecule inhibitor, 9-chloro-2-methylellipticinium acetate (CMEP), on the activation of AKT, its antiproliferation and apoptosis-inducing effects in prostate cancer cell lines: DU-145, PC-3, LNCaP, and CL-1, an androgen-independent LNCaP variant, and CL-1 xenograft mouse model were assessed by Western blot analysis, kinase assay, cell survival assay, and apoptosis assay in this report. It has been observed that the expression levels of AKT1, AKT2, and AKT3 vary, but the levels of phospho-Ser473 AKT and phospho-Thr308 AKT are quite unique in these cancer cell lines, and that CL-1 cells have the highest basal levels of AKT activation among these cell lines. In PC-3 cells, CMEP has been found to inhibit only AKT activation at both normal and serum-starvation conditions, not to inhibit PI3K, PDK1, or MAPK. More importantly, it has been discovered that CMEP inhibits cell proliferation, and induces apoptosis in prostate cancer cells which have high-levels of AKT activation and lack PTEN or harbor PTEN mutation, such as CL-1, LNCaP, and PC-3; only shows a minimal activity in DU-145 cancer cells which do not have AKT activation. Furthermore, it has been demonstrated that CMEP treatment inhibits phospho-Ser473 AKT and phospho-p70S6K while stimulating TSC2 in the tumor tissue from CL-1-bearing mice. In conclusion, by specific blockade of the activation of AKT, CMEP preferentially inhibits growth and induces apoptosis in prostate cancer cells which have high-levels of AKT activation.

Keywords: Abbreviations; AR; androgen receptor; CMEP; 9-chloro-2-methylellipticinium acetate; GSK-3; glycogen synthase kinase-3; MAPK; mitogen-activated kinases; mTOR; mammalian target of rapamycin; MTT; 3-[4,5-dimethythiazol-2-yl]2,5-diphenyltetrozolium bromide; PI3K; phosphatidylinositol 3-kinase; PKB; protein kinase B; PTEN; phosphatase and tensin homolog; TSC; tubererous sclerosis complexProstate cancer; AKT; Apoptosis; Small molecule

Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients by Malin Wickström; Katarina Danielsson; Linda Rickardson; Joachim Gullbo; Peter Nygren; Anders Isaksson; Rolf Larsson; Henrik Lövborg (pp. 25-33).

The thiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse. Disulfiram induces apoptosis in a number of tumor cell lines and was recently by us proposed to act as a 26S proteasome inhibitor. In this work we characterized disulfiram in vitro with regard to tumor-type specificity, possible mechanisms of action and drug resistance and cell death in human tumor cell lines and in 78 samples of tumor cells from patients using the fluorometric microculture cytotoxicity assay and the automated fluorescence-imaging microscope ArrayScan^®. Disulfiram induced cytotoxicity in a biphasic pattern in both cell lines and patient tumor cells. Disulfiram induced apoptosis as measured by cell membrane permeability, nuclear fragmentation/condensation and caspase-3/7 activation using high content screening assays. For many of the cell lines tested disulfiram was active in sub-micromolar concentrations. When comparing the logIC₅₀ patterns with other cytotoxic agents, disulfiram showed low correlation ( R<0.5) with all drugs except lactacystin ( R=0.69), a known proteasome inhibitor, indicating that the two substances may share mechanistic pathways. Disulfiram was more active in hematological than in solid tumor samples, but substantial activity was observed in carcinomas of the ovary and the breast and in non-small cell lung cancer. Disulfiram also displayed higher cytotoxic effect in cells from chronic lymphocytic leukemia than in normal lymphocytes ( p<0.05), which may indicate some tumor selectivity. These results together with large clinical experience and relatively mild side effects encourage clinical studies of disulfiram as an anti-cancer agent.

Keywords: Disulfiram; Cytotoxicity; Proteasome inhibitor; Patient tumor samples; Cell lines; Apoptosis

Identification of two biologically crucial hydroxyl groups of (−)-epigallocatechin gallate in osteoclast culture by Hiroshi Nakagawa; Keiji Hasumi; Masamichi Takami; Sachiko Aida-Hyugaji; Je-Tae Woo; Kazuo Nagai; Toshihisa Ishikawa; Masaaki Wachi (pp. 34-43).

(−)-Epigallocatechin gallate (EGCG) induces cell death of osteoclasts in an Fe²⁺- and H₂O₂-dependent manner. In the present study, we further explore the cytotoxic mechanism of EGCG using four EGCG analogues. Molecules methylated at position 4′ in the B ring (EGCG-4′- O-Me) or at position 4″ in the D-ring (EGCG-4″- O-Me) showed markedly decreased cytotoxicity to osteoclasts, indicating that hydroxyl groups at these two positions of EGCG are crucial for inducing cell death of osteoclasts. EGCG-4′- O-Me also showed the lowest Fe³⁺-reducing activity among five EGCGs. The Fe³⁺-reducing activity of EGCG was enhanced under conditions whereby protonated EGCG levels were increased, indicating that the protonated status of EGCG was involved in the Fe³⁺-reducing activity. The hydroxyl group at position 4″ in the D-ring was shown by quantum chemical calculation to be preferentially deprotonated among all of the hydroxyl groups in EGCGs. It was also shown that the highest occupied molecular orbital (HOMO) was localized to the B-ring of EGCGs, except for EGCG-4′- O-Me. We report here that the HOMO on the B-ring plays crucial roles in both the Fe³⁺-reducing activity of EGCG and the cytotoxicity of EGCG to osteoclasts, while deprotonation of the hydroxyl group at position 4″ in the D-ring plays a supplementary role.

Keywords: Abbreviations; CT; calcitonin; EGCG; (−)-epigallocatechin gallate; HOMO; highest occupied molecular orbital; LUMO; lowest unoccupied molecular orbital; OCL; osteoclast; PBS(−); PBS without Ca; ²⁺; and Mg; ²⁺; SAR; structure–activity relationship; SOD; superoxide dismutase; TRAP; tartrate-resistant acid phosphatase(−)-Epigallocatechin gallate; Ferric ion reduction; Hydrogen peroxide generation; Osteoclast; Quantum chemical calculation; SAR

Tezacitabine enhances the DNA-directed effects of fluoropyrimidines in human colon cancer cells and tumor xenografts by Pietro Taverna; Katherine Rendahl; Dragana Jekic-McMullen; Yi Shao; Kim Aardalen; Fernando Salangsang; Laura Doyle; Eddie Moler; Barbara Hibner (pp. 44-55).

Tezacitabine is a nucleoside analogue characterized by a dual mechanism of action. Following intracellular phosphorylation, the tezacitabine diphosphate irreversibly inhibits ribonucleotide reductase, while the tezacitabine triphosphate can be incorporated into DNA during replication or repair, resulting in DNA chain termination. In the present study we have investigated the effect of the combination of tezacitabine and 5-fluorouracil (5-FU) or 5-fluoro-2′-deoxyuridine (FUdR) on HCT 116 human colon carcinoma cells and xenografts. We used response surface analysis (RSA) and clonogenic assay to evaluate combination effects of tezacitabine and 5-FU. Tezacitabine is antagonistic when combined with 5-FU in the RSA assay and does not effect the clonogenicity of HCT 116 cells when compared with cells treated with 5-FU alone. However, when combined sequentially with FUdR, tezacitabine leads to potentiation of cell killing in the clonogenic assay, additivity in the RSA assay, and increased apoptosis when compared to FUdR alone, suggesting that cytotoxicity of fluoropyrimidines such as FUdR that have more DNA-directed effects can be potentiated by tezacitabine. We also report that oral administration of the fluoropyrimidine capecitabine, an oral prodrug of 5-FU, in combination with tezacitabine shows statistically significant additivity in the HCT 116 xenograft model. This interaction may be explained by the finding that tezacitabine elevates activity of thymidine phosphorylase (TP), the enzyme required for activation of the capecitabine prodrug in tumors. Our results provide evidence that tezacitabine enhances the DNA-directed effects of fluoropyrimidines in human colon cancer cells and may modulate the antitumor activity of fluoropyrimidines.

Keywords: Combination chemotherapy; Colon cancer; Tezacitabine; FMdC; Nucleoside analogs; Fluoropyrimidines

Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells by Tehila Tannin-Spitz; Shlomo Grossman; Sara Dovrat; Hugo E. Gottlieb; Margalit Bergman (pp. 56-67).

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER⁺ MCF-7 and ER⁻ MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G₂/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G₂ exit and initiation of mitosis, namely the p34^CDC2/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21^WAF, proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (ΔΨ) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells.

Keywords: Abbreviations; ER; estrogen receptor; FACS; fluorescence activated cell sorting; FCS; foetal calf serum; JC-1; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolocarbocyanin iodide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NMR; nuclear magnetic resonance; PI; propidium iodide; TLC; thin layer chromatographyApoptosis; Breast cancer; Cucurbitacin glucoside; F-actin; G; ₂; /M; p34; ^CDC2

Elevated gadd153/ chop expression during resveratrol-induced apoptosis in human colon cancer cells by Kyung Jin Woo; Tae Jin Lee; Sang Han Lee; Jin-Man Lee; Ji-Hyung Seo; Yong-Jin Jeong; Jong-Wook Park; Taeg Kyu Kwon (pp. 68-76).

Resveratrol (3,4′,5-tri-hydroxystilbene), a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. Resveratrol-induced dose-dependent apoptotic cell death in colon carcinoma cells, as measured by FACS analysis and internucleosomal DNA fragmentation assays. We demonstrate for the first time that resveratrol induce CCAAT/enhancer-binding protein-homologous protein (CHOP). Resveratrol-induced CHOP mRNA (and also protein) expression was inhibited by JNK specific inhibitor, but not ERK, p38 MAPK, PI3K and NF-κB inhibitors. Resveratrol-induced expression of CHOP involves the putative Sp1 site within the CHOP promoter region. Using a combination of the Sp1 cDNA transfection, the luciferase reporter assay and Sp1 inhibitor assay, we found that Sp1 site is required for resveratrol-mediated activation of the CHOP promoter. Suppression of CHOP expression by CHOP siRNA and treatment with mithramycin A attenuated resveratrol-induced apoptosis. Taken together, the present studies suggest that induction of CHOP protein may be involved, at least in part, in resveratrol-induced apoptosis.

Keywords: Resveratrol; CHOP; GADD153; Apoptosis; Sp1; Promoter; Colon cancer

Statins decrease TNF-α-induced osteoprotegerin production by endothelial cells and smooth muscle cells in vitro by E. Ben-Tal Cohen; P.J. Hohensinner; C. Kaun; G. Maurer; K. Huber; J. Wojta (pp. 77-83).

Recent reports have implicated osteoprotegerin (OPG) in cardiovascular disease processes. Endothelial and smooth muscle cells produce OPG and its expression in these cells is upregulated by inflammatory mediators. Statins, which besides their lipid lowering properties have various vasculoprotective effects, have been shown to regulate OPG expression in osteoblasts. We investigated whether statins affect the expression of OPG in human endothelial and smooth muscle cells. Using an ELISA we could demonstrate that statins reduce tumor necrosis factor-α (TNF-α)-induced OPG production in cultured human endothelial cells and smooth muscle cells. Atorvastatin also downregulated interleukin-1α (IL-1α)-induced OPG production in endothelial cells. A significant reduction of TNF-α-induced OPG was seen when statins were used in the nanomolar range. These results were confirmed at the level of specific mRNA expression by real-time-PCR. Using LDH leakage as a marker of cell damage we show that cell viability was not affected by statins at concentrations used in our study. The effect of statins on TNF-α-induced OPG production was reversed by mevalonate and geranyl–geranyl pyrophosphate at the level of protein production and at the level of mRNA expression, suggesting that it was brought about by inhibition of the mevalonic acid pathway and protein prenylation. Through our results we have added OPG to the list of molecules whose TNF-α-induced upregulation is counteracted by statins. If such an effect is also operative in the in vivo setting, one could postulate a role for statins in the modulation of cardiovascular disease processes possibly regulated by OPG.

Keywords: Osteoprotegerin; Statins; Endothelial cells; Inflammation

Evaluation of therapeutic potentials of site-specific PEGylated glucagon-like peptide-1 isomers as a type 2 anti-diabetic treatment: Insulinotropic activity, glucose-stabilizing capability, and proteolytic stability by Yu Seok Youn; Su Young Chae; Seulki Lee; Jae Eun Jeon; Hyong Goo Shin; Kang Choon Lee (pp. 84-93).

PEGylation has been considered to be a good biotechnique for improving the therapeutic value of glucagon-like peptide-1 (GLP-1) analogs for the treatment of type 2 diabetes. Despite the attractive anti-diabetic potentials, GLP-1 does not exert its full biological action because of its extremely short life-time in vivo due to rapid proteolytic degradation. Here, the enzyme-resistant mono-PEGylated GLP-1 isomers substituted at Lys²⁶- or Lys³⁴-amine were prepared through a newly devised site-specific PEGylation process using a maleic anhydride-protection/deprotection method. The therapeutic potentials of these site-specific PEGylated GLP-1 isomers (Lys²⁶- or Lys³⁴-PEG-GLP-1) along with His⁷-(N-terminus) PEG-GLP-1 were evaluated by examining their insulinotropic activity, glucose-stabilizing capability, and proteolytic stability. Lys³⁴-PEG-GLP-1 was found to have the well-preserved insulinotropic activity (93% efficacy versus GLP-1) in isolated rat pancreatic islets. Furthermore, Lys³⁴-PEG-GLP-1 showed the most prominent glucose-stabilizing capability, evaluated via an oral glucose tolerance test in db/db mice by considering the following three crucial factors: (i) maximum blood glucose level (BGL), (ii) required time to lower the BGL below 100mg/dl, and (iii) total hypoglycemic degree. Additionally, Lys³⁴-PEG-GLP-1 had longer half-lives than the other PEGylated GLP-1s in the dipeptidyl peptidase IV (DPP IV) inhibitor-treated liver or kidney homogenate, and its stability against DPP IV was also comparable to that of Lys²⁶-PEG-GLP-1. Taken together, Lys³⁴-PEG-GLP-1 displayed the promising characteristics in all evaluations versus His⁷- or Lys²⁶-PEG-GLP-1. This site-specific PEGylated GLP-1 analog would have therapeutic usefulness for treating type 2 diabetes on account of the well-preserved insulinotropic activity, the increased proteolytic stability, and thereby the improved glucose-stabilizing capability.

Keywords: Glucagons-like peptide-1; Type-2 diabetes; Dipeptidyl peptidase-IV; Neutral endopeptidase 24.11; PEGylation

Antagonism of the insulinotropic action of first generation imidazolines by openers of K_ATP channels by Antje Wienbergen; Claudia Bleck; Timm Grosse Lackmann; Ingo Rustenbeck (pp. 94-102).

The antagonism between K_ATP channel-blocking insulinotropic imidazolines – phentolamine, alinidine, idazoxan and efaroxan – and K_ATP channel openers, diazoxide and nucleoside diphosphates, was studied in mouse pancreatic islets and B-cells. In inside-out patches from B-cells, 500μM MgGDP abolished the inhibitory effect of the imidazolines. 300μM diazoxide further increased channel activity. The depolarizing effect of all imidazolines (100μM) on the B-cell membrane potential was practically completely antagonized by 300μM diazoxide. In contrast, diazoxide was unable to decrease the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which was elevated by phentolamine, whereas the [Ca²⁺]_i increases induced by the other imidazolines were promptly antagonized. The effects on [Ca²⁺]_i were reflected by the secretory activity in that the stimulatory effects of alinidine, idazoxan and efaroxan, but not that of phentolamine were antagonized by diazoxide. Metabolic inhibition of intact B-cells by 250μM NaCN, most likely by a decrease of the ATP/ADP ratio, significantly diminished the K_ATP channel-blocking effect of a low concentration of alinidine (10μM), whereas efaroxan proved to be susceptible even at a highly effective concentration (100μM). This may explain the oscillatory pattern of the [Ca²⁺]_i increase typically produced by efaroxan in pancreatic B-cells. In conclusion, the inhibitory effect of imidazolines on K_ATP channels, which is exerted at the pore-forming subunit, Kir6.2, is susceptible to the action of endogenous and exogenous K_ATP channel openers acting at the regulatory subunit SUR, which confers tissue specificity. With intact cells this antagonism can be obscured, possibly by intracellular accumulation of some imidazolines.

Keywords: Abbreviations; K; _ATP; channels; ATP-dependent K; ⁺; channels; [Ca; ²⁺; ]; cytosolic calcium concentrationPancreatic islets; B-cells; Cytosolic calcium concentration; K; _ATP; channel; Efaroxan; Imidazolines; Tolbutamide

Mechanisms for prostaglandin E₂ formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells by Fumiko Sekiguchi; Shino Saito; Kaori Takaoka; Hitomi Hayashi; Mami Nagataki; Keita Nagasawa; Hiroyuki Nishikawa; Hirofumi Matsui; Atsufumi Kawabata (pp. 103-114).

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E₂ (PGE₂) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE₂ release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE₂ formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE₂ formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE₂ formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE₂ formation in RGM1 cells.

Keywords: Abbreviations; AACOCF; ₃; arachidonyl trifluoromethyl ketone; ADAMs; a disintegrin and metalloproteinases; BEL; bromoenol lactone; COX; cyclooxygenase; cPLA; ₂; cytosolic Ca; ²⁺; -dependent phospholipase A; ₂; EGF; epidermal growth factor; EGFR-TK; EGF receptor-tyrosine kinase; ERK; extracellular signal-regulated kinase; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HB-EGF; heparin-binding-EGF; iPLA; ₂; Ca; ²⁺; -independent phospholipase A; ₂; JNK; c-Jun NH; ₂; -terminal kinase; MAPK; mitogen-activated protein kinase; MEK; MAPK/ERK kinase; MMP; matrix metalloproteinase; PAR; proteinase-activated receptor; PAR1-AP; PAR1-activating peptide; PGE; ₂; prostaglandin E; ₂; PI3-kinase; phosphatidyl inositol 3-kinase; PKC; protein kinase C; SLIGRL-NH; ₂; Ser-Leu-Ile-Gly-Arg-Leu-amide; TFLLR-NH; ₂; Thr-Phe-Leu-Leu-Arg-amide; TGF-α; transforming growth factor-αEGF receptor trans-activation; Cyclooxygenase-2 (COX-2); Heparin-binding-EGF; MAP kinases; Prostaglandin E; ₂; Proteinase-activated receptor-1 (PAR1)

Delineation of the motilin domain involved in desensitization and internalization of the motilin receptor by using full and partial antagonists by Anna Mitselos; Inge Depoortere; Theo L. Peeters (pp. 115-124).

Studies with fragments of the gastrointestinal peptide, motilin, indicate that the C-terminal region of this peptide plays an important role in the desensitization of the motilin receptor (MTLR).To verify this hypothesis we studied the desensitization, phosphorylation and internalization induced by motilin analogues of different chain length with agonistic and antagonistic properties in CHO–MTLR cells.We studied motilin [1–22], the [1–14] fragment, the analogues Phe³[1–22] and Phe³[1–14], and two putative antagonists, GM-109 and MA-2029 (modified 1–4 and 1–3 fragments). Activation and desensitization (2h preincubation with the motilin analogues 10μM) were studied in CHO–MTLR cells by an aequorin based luminescence assay. Phosphorylation was studied by immunoprecipitation and internalization was visualized in CHO–MTLR cells containing an enhanced green fluorescent protein (CHO–MTLR–EGFP).Motilin [1–22] and [1–14] were more potent than Phe³[1–22] and Phe³[1–14] (pEC₅₀: 9.77, 8.78, 7.36 and 6.65, respectively) to induce Ca²⁺ release. GM-109 and MA-2029 were without agonist activity. [1–22] and Phe³[1–22] decreased the second response to motilin from 78±2% to 11±3% and 34±3% ( P<0.001), respectively, whereas [1–14], Phe³[1–14], GM-109 and MA-2029 had no desensitizing effect (68±5%, 78±3%, 78±6% and 78±5%, respectively, P>0.05). The rank order of MTLR-phosphorylation was: [1–22]>[1–14]>Phe³[1–22]=Phe³[1–14]>GM-109=MA-2029. Only motilin [1–22] and [1–14] induced receptor MTLR–EGFP internalization as shown by a decrease in membrane fluorescence: 20±3% and 7±3%, respectively.The C-terminus of motilin enhances desensitization, phosphorylation and internalization of the MTLR while modifications of the N-terminus can favor a conformation of the receptor that is less susceptible to phosphorylation and internalization.

Keywords: Desensitization; Phosphorylation; Internalization; Motilin receptor; Motilin agonist; Motilin antagonist

Involvement of early growth response gene 1 in the modulation of microsomal prostaglandin E synthase 1 by epigallocatechin gallate in A549 human pulmonary epithelial cells by Yuseok Moon; Myoungjoo Lee; Hyun Yang (pp. 125-135).

The prostaglandin E₂ (PGE₂) can play critical roles in the pulmonary inflammation or carcinogenesis. It is the first investigation of the effect of a green tea polyphenol, (−)-epigallocatechin gallate (EGCG), on the PGE₂-producing microsomal prostaglandin E synthase 1 (mPGES-1) expression in the lung alveolar type II pneumocytes, A549 cells as an epithelial model. EGCG enhanced cyclooxygenase (COX)-2 and mPGES-1 gene expression as well as PGE₂. Among several tea catechins, EGCG was most effective in inducing mPGES-1 expression. Moreover, even in the cytokine-stimulated cells, mPGES-1 protein was super-induced by EGCG treatment. As signaling mediators in mPGES-1 induction by EGCG, active ERK1/2 MAP kinases and early growth response gene 1 (EGR-1) were increased after exposure to EGCG. Moreover, EGCG stimulated the nuclear translocation of the EGR-1 protein in A549 cells through ERK signaling pathway. Recent studies demonstrate that EGR-1 is a key transcription factor in mPGES-1 gene expression. When blocking the gene expression of EGR-1 with EGR-1 siRNA or ERK inhibitor, EGCG-induced mPGES-1 was suppressed in both cases. mPGES-1 promoter with deleted or point-mutated EGR-1 binding sites showed significantly less response to the EGCG stimulation, which also implicated the importance of EGR-1 binding in promoting mPGES-1 gene expression. Taken all, EGCG was strong inducer of EGR-1 expression and mediated EGR-1 nuclear translocation via ERK signaling pathway in A549 pulmonary epithelial cells. Induced EGR-1 then stimulated the induction of mPGES-1 gene expression and this effect mechanistically can be linked to the pharmacological or toxicological actions after human exposure to green tea catechins.

Keywords: Abbreviations; EGCG; (−)-epigallocatechin gallate; PGES; prostaglandin E synthase; COX; cyclooxygenase; EGR-1; early growth response gene 1; cPGES; cytosolic PGES; mPGES; microsomal PGES; NSCLC; non-small cell lung cancer; MAP kinase; mitogen activated protein kinase; ERK; extracellular signal-regulated protein kinase; PDGF; platlet derived growth factor; EGF; epithermal growth factor; NF-κB; nuclear factor κB; AP-1; activator protein-1; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2; H; -tetrazolium; TBST; Tris-buffered saline plus Tween 0.05%; RT-PCR; reverse transcription-polymerase chain reaction; PGH; ₂; prostaglandin H; ₂; PGE; ₂; prostaglandin E; ₂; IRAK; interleukin-1β receptor-associated kinase; CREBP; cyclic AMP response element binding proteinMicrosomal prostaglandin E synthase 1; Epigallocatechin gallate; Early growth response gene 1; Pulmonary inflammation; ERK1/2; Cyclooxygenase 2

Triptolide, a diterpenoid triepoxide, suppresses inflammation and cartilage destruction in collagen-induced arthritis mice by Na Lin; Chunfang Liu; Cheng Xiao; Hongwei Jia; Keisuke Imada; Hao Wu; Akira Ito (pp. 136-146).

Chinese herbal remedy Tripterygium wilfordii Hook. f. (TWHF) has been reported to be therapeutically efficacious in the treatment of rheumatoid arthritis (RA), but its in vivo actions have not been clarified. The purpose of this study was to investigate the effects of triptolide, a diterpenoid triepoxide extracted from TWHF, on inflammation and cartilage destruction in collagen-induced arthritis (CIA) model mice. Histological examination demonstrated that triptolide significantly reduced the inflammatory responses and cartilage damage in the joint tissues. Interestingly, triptolide interfered with CIA-augmented expression of matrix metalloproteinases-13 and -3, which are considered to be key enzymes in the pathological destruction of cartilage, and simultaneously augmented CIA-reduced tissue inhibitors of metalloproteinases-1 and -2 expression in the joints. Moreover, triptolide inhibited prostaglandin E₂ production via selective suppression of the production and gene expression of cyclooxygenase (COX)-2, but not COX-1. The levels of interleukin (IL)-1β, tumor necrosis factor α and IL-6 were also decreased by triptolide in the joint tissues and sera as well as the suppression of CIA-mediated expression of their mRNAs in the joints. In addition, triptolide treatment in vivo was able to reduce an abundance of nuclear factor-κB, the transcriptional factor closely related to the inflammatory process, in articular cartilage and synovium in CIA mice. These results suggest that triptolide exerts novel chondroprotective and anti-inflammatory effects on RA, and the therapeutic action of TWHF on RA is, in part, due to the triptolide activities.

Keywords: Abbreviations; CIA; collagen-induced arthritis; CII; type II collagen; COX; cyclooxygenase; DAB; diaminobenzidine; DMARDs; disease modifying antirheumatic drugs; IL; interleukin; MMPs; matrix metalloproteinases; NF; nuclear factor; PBS; phosphate buffered saline; PG; prostaglandin; quantitative real-time RT-PCR; quantitative real-time reverse transcriptase-polymerase chain reaction; RA; rheumatoid arthritis; RIA; radio immunoassay; SABC; streptavidin–biotin complex; TIMPs; tissue inhibitors of metalloproteinases; TNF; tumor necrosis factor; TWHF; Tripterygium wilfordii; Hook. f.Triptolide; Inflammation; Matrix metalloproteinase; Cyclooxygenase; Inflammatory cytokines; Cartilage destruction; Collagen-induced arthritis mice

Evidence for significant contribution of a newly identified monoamine transporter (PMAT) to serotonin uptake in the human brain by Mingyan Zhou; Karen Engel; Joanne Wang (pp. 147-154).

The high affinity serotonin transporter (SERT) constitutes the principal pathway for removal of serotonin (5-HT) from extracellular fluid of brain, but evidence indicates that other transporters may also be involved in this process. We recently reported the cloning of a novel plasma membrane monoamine transporter (PMAT), which is abundantly expressed in the human brain and avidly transports 5-HT [Engel K, Zhou M, Wang J. Identification and characterization of a novel monoamine transporter in the human brain. J Biol Chem 2004;279:50042–9]. In this study, we evaluated whether PMAT contributes to total human brain uptake of 5-HT using a hybrid depletion approach in Xenopus laevis oocytes. We also examined whether PMAT interacts with selective serotonin reuptake inhibitors (SSRIs) using MDCK cells stably expressing recombinant human PMAT. Microinjection of total human brain poly(A)⁺ mRNA into oocytes elicited ∼2.5–3-fold increase in 5-HT uptake. Pre-hybridization of poly(A)⁺ mRNA with PMAT or SERT antisense oligonucleotides significantly reduced mRNA-induced 5-HT uptake. An additive inhibitory effect was observed when poly(A)⁺ mRNA was co-hybridized with both PMAT and SERT antisense oligonucleotides. In contrast, mRNA-induced 5-HT uptake was not affected by pre-hybridization with sense oligonucleotides. These data suggest that functional transcripts of PMAT are present in the human brain, and the PMAT transporter may be significantly involved in brain uptake of 5-HT. All five tested SSRIs inhibited PMAT with IC₅₀ values ranging from 11 to 116μM, which are much greater than clinically encountered concentrations, suggesting that PMAT activity is minimally affected by SSRI therapies.

Keywords: Abbreviations; PMAT; plasma membrane monoamine transporter; 5-HT; 5-hydroytryptamine; SERT; serotonin transporter; SSRI; selective serotonin reuptake inhibitor; CNS; central nervous system; MDCK; Madin–Darby canine kidneyPMAT; hENT4; Serotonin; Hybrid depletion; Human brain; SSRIs

Obesity-induced increase of CYP2E1 activity and its effect on disposition kinetics of chlorzoxazone in Zucker rats by Phisit Khemawoot; Koichi Yokogawa; Tsutomu Shimada; Ken-ichi Miyamoto (pp. 155-162).

This study was designed to investigate the induction of CYP2E1 in obese Zucker rats and its effect on the disposition kinetics of chlorzoxazone (CZX). CZX 20mg/kg was administered to three groups of rats: normal Zucker rats fed a normal diet (ND), normal Zucker rats fed a high-fat diet (HF), and genetically obese Zucker rats fed a normal diet (OB). The values of the area under the plasma concentration–time curve from 0 to ∞ (AUC_∞) of CZX were in the order of ND>HF>OB rats. The AUC_∞ values of total 6-hydroxychlorzoxazone (6OHCZX-T), which is considered to be a CYP2E1 metabolic marker, were in the opposite order. The values of the AUC_∞ ratio (6OHCZX–T/CZX) in ND, HF and OB rats were approximately 0.2, 0.3 and 0.4, respectively. The CZX concentration in fat was much higher than the concentrations in plasma, liver and kidney in all groups. Induction of CYP2E1 protein was greater in both liver and fat of OB rats than in those of HF rats. Microsomal activity of CYP2E1 in liver and fat was also in the order of OB>HF>NM rats. These results suggest that CYP2E1 may be induced in liver and fat of obese patients, thereby potentially altering the disposition kinetics of not only CZX, but also other lipophilic drugs metabolized by CYP2E1.

Keywords: Abbreviations; 6OHCZX; 6-hydroxychlorzoxazone; 6OHCZX-G; glucuronidated 6-hydroxychlorzoxazone; 6OHCZX-T; total 6-hydroxychlorzoxazone; AST; aspartate aminotransferase; CYP; cytochrome P450; CZX; chlorzoxazone; CZX-G; glucuronidated chlorzoxazone; PNP; p; -nitrophenol; UGT; UDP-glucuronosyltransferaseObesity; CYP2E1; Chlorzoxazone; Zucker rat

Erratum to “Proteasome-independent down-regulation of estrogen receptor-alpha (ERalpha) in breast cancer cells treated with 4,4′-dihydroxy- trans-stilbene�? [Biochem. Pharmacol. 72 (5) (2006) 573–581] by Kannan V. Balan; Zhiyong Han (pp. 163-164).

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Biochemical Pharmacology (v.73, #1)