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Biochemical Pharmacology (v.74, #1)

Editorial Advisory Board (pp. iii).

Calcium channel antagonists: Clinical uses—Past, present and future by David J. Triggle (pp. 1-9).

The calcium channel antagonists are a mature group of drugs directed at cardiovascular diseases including hypertension, angina, peripheral vascular disorders and some arrhythmic conditions. Their sites and mechanisms of actions have been well explored over the past two decades and their interactions at the α₁ subunit of L-type channels (Ca_V1.1–1.4) have made them valuable molecular tools for channel classification and localization. With the realization that other members of the voltage-gated calcium channel family exist – Ca_V2.1–2.3 and Ca_V3.1–3.3 – considerable effort has been directed to drug discovery at these channel types where therapeutic prospects exist for a variety of disorders including pain, epilepsy, affective disorders, neurodegenerative disorders, etc. In contrast to the situation with the L-type channel antagonists success in developing small molecule antagonists of therapeutic utility for these other channel types has thus far been lacking. The reasons for this are explored and potential new directions are indicated including male fertility, bone growth, immune disorders, cancer and schistosomiasis.

Keywords: Calcium channel antagonists; 1,4-Dihydropyridines; Nifedipine; Verapamil; Diltiazem; Pain; Stroke; Contraception; Bone formation; Fertility’ immune cells; T cells; Schistosomiasis

Quinolone analogue inhibits tubulin polymerization and induces apoptosis via Cdk1-involved signaling pathways by Ying-Cheng Chen; Pin-Hsuan Lu; Shiow-Lin Pan; Che-Ming Teng; Sheng-Chu Kuo; Tsung-Ping Lin; Yunn-Fang Ho; Yu-Chun Huang; Jih-Hwa Guh (pp. 10-19).

Cancer chemotherapeutic agents that interfere with tubulin/microtubule function are in extensive use. Quinolone is a common structure in alkaloids and its related components exhibit several pharmacological activities. In this study, we have identified the anticancer mechanisms of 2-phenyl-4-quinolone. 2-Phenyl-4-quinolone displayed anti-proliferative effect in several cancer types, including hormone-resistant prostate cancer PC-3, hepatocellular carcinoma Hep3B and HepG2, non-small cell lung cancer A549 and P-glycoprotein-rich breast cancer NCI/ADR-RES cells. The IC₅₀ values were 0.85, 1.81, 3.32, 0.90 and 1.53μM, respectively. 2-Phenyl-4-quinolone caused G2/M arrest of the cell-cycle and a subsequent apoptosis. The turbidity assay showed an inhibitory effect on tubulin polymerization. After immunochemical examination, the data demonstrated that the microtubules were arranged irregularly into dipolarity showing prometaphase-like states. Furthermore, 2-Phenyl-4-quinolone induced the Mcl-1 cleavage, the phosphorylation of Bcl-2 and Bcl-xL (12-h treatment), and the caspase activation including caspase-8, -2 and -3 (24-h treatment). The exposure of cells to 2-phenyl-4-quinolone caused Cdk1 activation by several observations, namely (i) elevation of cyclin B1 expression, (ii) dephosphorylation on inhibitory Tyr-15 of Cdk1, and (iii) dephosphorylation on Ser-216 of Cdc25c. Moreover, a long-term treatment (36h) caused the release reaction and subsequent nuclear translocation of AIF. In summary, it is suggested that 2-phenyl-4-quinolone displays anticancer effect through the dysregulation of mitotic spindles and induction of mitotic arrest. Furthermore, participation of cell-cycle regulators, Bcl-2 family of proteins, activation of caspases and release of AIF may mutually cross-regulate the apoptotic signaling cascades induced by 2-phenyl-4-quinolone.

Keywords: Quinolone; Mitotic arrest; Cdk1; Bcl-2 family of proteins; Caspase; AIF

Accumulation, platinum–DNA adduct formation and cytotoxicity of cisplatin, oxaliplatin and satraplatin in sensitive and resistant human osteosarcoma cell lines, characterized by p53 wild-type status by Laura Martelli; Francesco Di Mario; Paolo Botti; Eugenio Ragazzi; Mario Martelli; Lloyd Kelland (pp. 20-27).

p53 gene status is implicated in the cytotoxic drug sensitivity and published research has been mostly addressed to cisplatin (CDDP) activity. Previous study in our laboratory considered p53 mutant cell lines A431 (parental) and A431/Pt (CDDP-resistant counterpart, resistance factor R.F.=2.6). For a comparison which contributes to a deeper appreciation of the process that mediates the Pt drug cellular effects, we extended our investigation to the p53 wild-type cell lines U2-OS (human osteosarcoma) and its CDDP-resistant counterpart U2-OS/Pt (R.F.=5). We compared the activity of CDDP, oxaliplatin (L-OHP) and satraplatin (JM216) whose hydrophobicity rank is JM216>L-OHP>CDDP.In U2-OS cells the three drugs accumulated similarly, while in U2-OS/Pt the most hydrophobic drugs were privileged. No significant differences in efflux were observed between sensitive and resistant cell lines. The growing of CDDP resistance seems to be overcome by increasing the hydrophobicity of the Pt agent. An almost linear trend seems to relate R.F. and drug hydrophobicity in U2-OS/Pt and A431/Pt cells. DNA platination in U2-OS as in A431 cells is at the lowest levels for L-OHP. In U2-OS cell line the IC₅₀ of CDDP (17.6μM) and JM216 (88.02μM) do not correlate with their similar levels of Pt–DNA adducts (mean value ∼0.14pmolPt/μg DNA). The presence of a wild-type p53 exalts either CDDP cytotoxicity (two-fold more active in U2-OS than in A431 cells) and CDDP resistance in comparison to a p53 mutant type. The p53 status seems to not improve JM216 or L-OHP cytotoxicity in both cell lines.

Keywords: U2-OS human osteosarcoma cell line; Pt drugs; Drug resistance; Drug accumulation; Pt–DNA binding; Hydrophobicity; Cell sensitivity; p53

New platinum(II) complexes containing both an O, O′-chelated acetylacetonate ligand and a sulfur ligand in the platinum coordination sphere induce apoptosis in HeLa cervical carcinoma cells by Antonella Muscella; Nadia Calabriso; Sandra A. De Pascali; Loredana Urso; Antonella Ciccarese; Francesco P. Fanizzi; Danilo Migoni; Santo Marsigliante (pp. 28-40).

We report the cytotoxic effects obtained in HeLa cells of three newly synthesized platinum complexes containing both an O, O′-chelated acetylacetonate ligand and a sulfur ligand in the platinum coordination sphere, which show, by¹H NMR, negligible reactivity with purine bases. These compounds induce cell death with [Pt( O, O′-acac)(γ-acac)(DMS)] being the most effective (IC₅₀=0.98±0.056 and 1.82±0.023μM for [Pt( O, O′-acac)(γ-acac)(DMS)] and cisplatin, respectively). About 50% of cells died after 5h treatment with 100μM [Pt( O, O′-acac)(γ-acac)(DMS)] whilst a 16h incubation was required to get the same results using 100μM cisplatin. Cellular accumulation measurements, after treatment with equimolar drug concentrations, indicated the major lipophilicity and cellular uptake of the new compounds. While the cytotoxicity of cisplatin was due to both intracellular accumulation and DNA binding, that of [Pt( O, O′-acac)(γ-acac)(DMS)] was associated with intracellular Pt accumulation only, since it has low reactivity to DNA in intact cells and in vitro. The reaction of the new complexes with guanosine and 5′-GMP was negligible, whereas thel-methionine instantly reacted with the initial Pt complexes. Both cisplatin and [Pt( O, O′-acac)(γ-acac)(DMS)] induced apoptosis in HeLa cells. [Pt( O, O′-acac)(γ-acac)(DMS)] provoked the early signs of apoptosis induction (cleavage of PARP and activation of caspases-9, -3 and -7) only 1h after addition of the drug. However, in cisplatin-treated cells, cleavage of PARP was seen after 9h with activation of caspases also proceeding more slowly. In conclusion, these results indicate that the newly synthesized platinum(II) complexes have high and rapid cytotoxic activity in vitro, and suggest that DNA may not be their primary target.

Keywords: Cisplatin; HeLa; Apoptosis; ERK

C421 allele-specific ABCG2 gene amplification confers resistance to the antitumor triazoloacridone C-1305 in human lung cancer cells by Eran E. Bram; Ilan Ifergan; Michal Grimberg; Krzysztof Lemke; Andrzej Skladanowski; Yehuda G. Assaraf (pp. 41-53).

The A421 ABCG2 genotype is a frequent polymorphism encoding the K141 transporter, which is associated with a significant decrease in transporter expression and function when compared to the wild type (wt) C421 allele encoding the Q141 ABCG2. Here we show that during the acquisition of resistance to the novel triazoloacridone antitumor agent C-1305 in lung cancer cells harboring a heterozygous C421A genotype, a marked C421 allele-specific ABCG2 gene amplification occurred. This monoallelic C421 ABCG2 gene amplification brought about the overexpression of both C421 ABCG2 mRNA and the transporter at the plasma membrane. This resulted in the lack of cellular drug accumulation due to increased efflux of both C1305 and C-1311, a fluorescent imidazoacridone homologue of C-1305, as well as marked resistance to these antitumor agents and to established ABCG2 substrates including mitoxantrone and SN-38. Consistently, the accumulation and sensitivity to these drugs were restored upon incubation with the potent and specific ABCG2 transport inhibitors Ko143 and fumitremorgin C. Moreover, upon transfection into HEK293 cells, the wt Q141 ABCG2 allele displayed a significantly decreased accumulation of C-1311 and increased resistance to C-1305, C-1311 and mitoxantrone, when compared to the K141 ABCG2 transfectant. Hence, the current study provides the first evidence that during the exposure to anticancer drugs, an allele-specific Q141 ABCG2 gene amplification occurs that confers a drug resistance advantage when compared to the K141 ABCG2. These findings have important implications for the selection and expansion of malignant anticancer drug resistant clones during chemotherapy with ABCG2 drugs.

Keywords: Abbreviations; MDR; multidrug resistance; ABC; ATP-binding cassette; BCRP; breast cancer resistance protein; MRP; multidrug resistance protein; Pgp; P-glycoproteinChemotherapy; ABC transporter; Efflux pumps; Drug resistance

Lycopene binds PDGF-BB and inhibits PDGF-BB-induced intracellular signaling transduction pathway in rat smooth muscle cells by Huey-Ming Lo; Chi-Feng Hung; Yu-Lun Tseng; Bing-Huei Chen; Jr-Shian Jian; Wen-Bin Wu (pp. 54-63).

Cardiovascular diseases (CVDs) result from the sub-endothelial accumulation of inflammatory cells and smooth muscle cells (SMCs). Lycopene, a natural compound from tomato, has been suggested to play a role in CVD prevention. However, its action mechanism is still largely unknown. In this study, we examined the effect of lycopene on SMCs. We found that preincubation of PDGF-BB with lycopene resulted in a marked inhibition on PDGF-BB-induced PDGF receptor-β (PDGFR-β), PLCγ, and ERK1/2 phosphorylation in rat A10 SMCs and primary cultured aortic SMCs. In striking contrast, lycopene did not influence EGF-induced ERK1/2 phosphorylation. Surprisingly, further analysis indicates that lycopene could directly bind PDGF-BB and inhibit PDGF-BB-SMC interaction, as determined by dot binding assay and Western blotting. In functional studies, lycopene inhibited PDGF-BB-induced SMC proliferation and migration toward gelatin and collagen at concentrations ranging from 2 to 10μM. On the contrary, lycopene did not inhibit bFGF- and VEGF-induced endothelial cell migration. Gelatin zymography demonstrated that lycopene's effect on SMC migration was not due to the inhibition of matrix metalloproteinases (MMPs). Taken together, our results provide the first evidence showing that lycopene inhibits PDGF-BB-induced signaling, proliferation and migration in rat A10 and aortic SMCs. One of the action mechanisms is that lycopene is capable of binding PDGF-BB and inhibiting its interaction with SMC, which is quite different from those previously developed PDGFR-β antagonists. The results presented here may help us to better understand the beneficial effects of lycopene in CVD prevention.

Keywords: Abbreviations; Ab; antibody; EGCG; (−)-epigallocatechin-3-gallate; mAb; monoclonal antibody; MMP; matrix metalloproteinase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; EGF; epidermal growth factor; ERK; extracellular matrix-regulated kinase; PDGF; platelet-derived growth factor; PDGFR-β; PDGF receptor β; SMC(s); smooth muscle cell(s)Cardiovascular disease; Lycopene; Natural compound; PDGF; Signaling pathway; Tomato

Agonist-stimulated reactive oxygen species formation regulates β2-adrenergic receptor signal transduction by Nader H. Moniri; Yehia Daaka (pp. 64-73).

Generation of reactive oxygen species (ROS) can occur upon agonist stimulation of surface receptors to modulate downstream signaling processes. Here, we show that activation of the β2 adrenergic receptor (β2AR) by stimulation with the agonist isoproterenol leads to generation of ROS that is required for β2AR signal transduction. Specifically, we show that inhibition of NADPH oxidase with diphenyliodonium chloride, inhibition of the small GTPase Rac1 with NSC23766, and inhibition of formed ROS with the antioxidant N-acetyl-l-cysteine decreases β2AR-mediated cAMP formation, protein kinase A activation, and receptor phosphorylation and internalization, but does not impact ligand binding. The results also show that inhibition of ROS attenuates active β2AR-mediated binding of GTP to α subunits of heterotrimeric G proteins. Based on these results, we propose that agonist-dependent ROS formation is needed for β2AR signal transduction, perhaps through stabilization of active receptor conformers by redox-mediated modification of receptor and/or Gα proteins cysteine residues.

Keywords: β2-Adrenergic receptor; Reactive oxygen species; G-proteins; Cyclic AMP; Inositol phosphates; Receptor internalization

Depolarization and decreased surface expression of K⁺ channels contribute to NSAID-inhibition of intestinal restitution by L.C. Freeman; D.F. Narvaez; A. McCoy; F.B. von Stein; S. Young; K. Silver; S. Ganta; D. Koch; R. Hunter; R.F. Gilmour; J.D. Lillich (pp. 74-85).

Non-steroidal anti-inflammatory drugs (NSAIDs) contribute to gastrointestinal ulcer formation by inhibiting epithelial cell migration and mucosal restitution; however, the drug-affected signaling pathways are poorly defined. We investigated whether NSAID inhibition of intestinal epithelial migration is associated with depletion of intracellular polyamines, depolarization of membrane potential ( E_m) and altered surface expression of K⁺ channels. Epithelial cell migration in response to the wounding of confluent IEC-6 and IEC-Cdx2 monolayers was reduced by indomethacin (100μM), phenylbutazone (100μM) and NS-398 (100μM) but not by SC-560 (1μM). NSAID-inhibition of intestinal cell migration was not associated with depletion of intracellular polyamines. Treatment of IEC-6 and IEC-Cdx2 cells with indomethacin, phenylbutazone and NS-398 induced significant depolarization of E_m, whereas treatment with SC-560 had no effect on E_m. The E_m of IEC-Cdx2 cells was: −38.5±1.8mV under control conditions; −35.9±1.6mV after treatment with SC-560; −18.8±1.2mV after treatment with indomethacin; and −23.7±1.4mV after treatment with NS-398. Whereas SC-560 had no significant effects on the total cellular expression of K_v1.4 channel protein, indomethacin and NS-398 decreased not only the total cellular expression of K_v1.4, but also the cell surface expression of both K_v1.4 and K_v1.6 channel subunits in IEC-Cdx2. Both K_v1.4 and K_v1.6 channel proteins were immunoprecipitated by K_v1.4 antibody from IEC-Cdx2 lysates, indicating that these subunits co-assemble to form heteromeric K_v channels. These results suggest that NSAID inhibition of epithelial cell migration is independent of polyamine-depletion, and is associated with depolarization of E_m and decreased surface expression of heteromeric K_v1 channels.

Keywords: Non-steroidal anti-inflammatory drugs; Intestinal epithelial cells; Membrane potential; Potassium channels

Outer membrane protein A of Acinetobacter baumannii induces differentiation of CD4⁺ T cells toward a Th1 polarizing phenotype through the activation of dendritic cells by Jun Sik Lee; Je Chul Lee; Chang-Min Lee; In Duk Jung; Young-Il Jeong; Eun-Young Seong; Hae-Young Chung; Yeong-Min Park (pp. 86-97).

Acinetobacter baumannii is an increasing hospital-acquired pathogen that causes a various type of infections, but little is known about the protective immune response to this microorganism. Outer membrane protein A of A. baumannii (AbOmpA) is a major porin protein and plays an important role in pathogenesis. We analyzed interaction between AbOmpA and dendritic cells (DCs) to characterize the role of this protein in promoting innate and adaptive immune responses. AbOmpA functionally activates bone marrow-derived DCs by augmenting expression of the surface markers, CD40, CD54, B7 family (CD80 and CD86) and major histocompatibility complex class I and II. AbOmpA induces production of Th1-promoting interleukin-12 from DCs and augments the syngeneic and allogeneic immunostimulatory capacity of DCs. AbOmpA stimulates production of interferon-γ from T cells in mixed lymphocyte reactions, which suggesting Th1-polarizing capacity. CD4⁺ T cells stimulated by AbOmpA-stimulated DCs show a Th1-polarizing cytokine profile. The expression of surface markers on DCs is mediated by both mitogen-activated protein kinases and NF-κB pathways. Our findings suggest that AbOmpA induces maturation of DCs and drives Th1 polarization, which are important properties for determining the nature of immune response against A. baumannii.

Keywords: Dendritic cells; Outer membrane protein A; MAP kinases; Th1 cells; Interleukin-12; Interferon-γ

The determination and correlation of molecular and cellular equilibrium K_d and kinetic k_off values for small molecule allosteric antagonists of LFA-1 by Gary O. Caviness; Mark E. Labadia; Patricia A. Giblin; Joseph R. Woska Jr.; Kathleen Last-Barney; Deborah D. Jeanfavre; Maurice M. Morelock (pp. 98-106).

Molecular K_d and k_off parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1). The SPR assay involves using the non-blocking mAb TS2/4 to immobilize full-length LFA-1 on a hydrophilic chip surface, and the soluble, native ligand sICAM-1 to probe the fraction of free LFA-1. To determine the fraction of free LFA-1 on cell surfaces, a flow cytometry assay was developed utilizing the fluorophore-labeled Fab R3.1. The R3.1 antibody has been previously demonstrated to block the ability of both ICAM-1 and antagonists to bind to purified and cell-surface LFA-1. The molecular and ex vivo cellular parameters were determined for a set of nine structurally-related LFA-1 allosteric antagonists. The relationships between the parameters determined in the ELISA ( K_d), SPR ( k_off), and flow cytometry ( k_off) assays were shown to be linear with slopes approximately equal to 1, and a correlation analysis showed that the three assay datasets were equivalent at the α=0.05 level. These results were unexpected, as the ELISA and SPR assays involve high affinity LFA-1, and the flow cytometry assays involve cell surface LFA-1 in whole-blood, in which a distribution of affinity states would be expected. Nevertheless, the results presented herein show that the K_d and k_off's determined in molecular assays can be used as predictors of LFA-1 receptor occupancy in ex vivo assays.

Keywords: SPR; Flow-cytometry; Receptor-occupancy; Allosteric antagonists; Off-rates; Adhesion molecules

PGD₂ metabolism in plasma: Kinetics and relationship with bioactivity on DP1 and CRTH2 receptors by Rufina Schuligoi; Ronald Schmidt; Gerd Geisslinger; Manfred Kollroser; Bernhard A. Peskar; Akos Heinemann (pp. 107-117).

Prostaglandin (PG)D₂, an important mediator in allergic diseases, is rapidly transformed in plasma to active metabolites that bind and activate two distinct receptors, DP1 and CRTH2. Since the rate of PGD₂ degradation and the bioactivity of the resulting metabolites are still unclear, the aim of our study was to analyze the kinetics and biological effects of PGD₂ metabolites formed in plasma. Eosinophil shape change was taken as a parameter of chemotactic activation mediated by CRTH2 whereas inhibition of platelet aggregation served as a measure of DP1 activity. PGD₂ was degraded in plasma with an apparent half-life of approximately 30min, accompanied by a loss of potency in inhibiting platelet aggregation as well as inducing eosinophil stimulation. Incubation of PGD₂ in plasma for 120min caused an increase in the IC₅₀ for platelet aggregation by a factor of 6.5 and an increase of the EC₅₀ for eosinophil shape change by a factor of 7.2. However, tandem mass spectrometry analysis showed that incubation of PGD₂ in plasma for 120min resulted in clearance of PGD₂ of more than 92%, which was mirrored by a continuous formation of Δ¹²-PGD₂ and Δ¹²-PGJ₂, whereas only small amounts of 15d-PGD₂ and 15d-PGJ₂ were detected. Interestingly, a rapid degradation of PGD₂ was also observed in serum, which was not prevented by pepsin digestion of serum preceding the addition of PGD₂. Therefore, despite extensive non-enzymatic metabolization of PGD₂ in plasma, its biological activity with respect to DP1 and CRTH2 is maintained through the formation of bioactive metabolites.

Keywords: PGD2; PGD2 metabolites; Eosinophils; Chemotaxis; Platelet aggregation; PGD2 receptors DP1 and CRTH2

Guggulsterone inhibits tumor cell proliferation, induces S-phase arrest, and promotes apoptosis through activation of c-Jun N-terminal kinase, suppression of Akt pathway, and downregulation of antiapoptotic gene products by Shishir Shishodia; Gautam Sethi; Kwang Seok Ahn; Bharat B. Aggarwal (pp. 118-130).

Guggulsterone is a plant polyphenol traditionally used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, possibly through an anti-inflammatory mechanism. Whether this steroid has any role in cancer is not known. In this study, we found that guggulsterone inhibits the proliferation of wide variety of human tumor cell types including leukemia, head and neck carcinoma, multiple myeloma, lung carcinoma, melanoma, breast carcinoma, and ovarian carcinoma. Guggulsterone also inhibited the proliferation of drug-resistant cancer cells (e.g., gleevac-resistant leukemia, dexamethasone-resistant multiple myeloma, and doxorubicin-resistant breast cancer cells). Guggulsterone suppressed the proliferation of cells through inhibition of DNA synthesis, producing cell cycle arrest in S-phase, and this arrest correlated with a decrease in the levels of cyclin D1 and cdc2 and a concomitant increase in the levels of cyclin-dependent kinase inhibitor p21 and p27. Guggulsterone-induced apoptosis as indicated by increase in the number of Annexin V- and TUNEL-positive cells, through the downregulation of anti-apoptototic products. The apoptosis induced by guggulsterone was also indicated by the activation of caspase-8, bid cleavage, cytochrome c release, caspase-9 activation, caspase-3 activation, and PARP cleavage. The apoptotic effects of guggulsterone were preceded by activation of JNK and downregulation of Akt activity. JNK was needed for guggulsterone-induced apoptosis, inasmuch as inhibition of JNK by pharmacological inhibitors or by genetic deletion of MKK4 (activator of JNK) abolished the activity. Overall, our results indicate that guggulsterone can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and downregulation of antiapoptotic protein expression.

Keywords: Abbreviations; NF-κB; nuclear factor-kappa B; COX-2; cyclooxygenase-2; MMP-9; matrix metalloproteinase-9; FBS; fetal bovine serum; SDS; sodium dodecyl sulfate; TUNEL; terminal deoxyuridine triphosphate nick end-labeling; JNK; cJun N-terminal kinase; MKK; mitogen-activated protein kinase kinase; PARP; poly (ADP-ribose) polymerase activationGuggulsterone; JNK; Caspase; Akt; Apoptosis

Specific blockage of ligand-induced degradation of the Ah receptor by proteasome but not calpain inhibitors in cell culture lines from different species by Richard S. Pollenz (pp. 131-143).

To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75μM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1–5μM), proteasome inhibitor I (5–10μM) or lactacystin (5–15μM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

Keywords: Abbreviations; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; AHR; aryl hydrocarbon receptor; ARNT; Ah receptor nuclear translocator; GAR-HRP; goat anti-rabbit antibodies conjugated to horseradish peroxidase; GAR-RHO; goat anti-rabbit antibodies conjugated to rhodamine; t; -BOC-leu-met; t; -BOC-; l; -leucyl-; l; -methionine amideAryl hydrocarbon receptor; Calpain; Proteasome; Protein degradation; TCDD; Ubiquitin

Fumonisin B₁ exposure and its selective effect on porcine jejunal segment: Sphingolipids, glycolipids and trans-epithelial passage disturbance by Nicolas Loiseau; Laurent Debrauwer; Tounkang Sambou; Sandrine Bouhet; J. David Miller; Pascal G. Martin; Jean-Luc Viadère; Philippe Pinton; Olivier Puel; Thierry Pineau; Jacques Tulliez; Pierre Galtier; Isabelle P. Oswald (pp. 144-152).

Fumonisin B₁ (FB₁) is a mycotoxin produced by Fusarium verticillioides, the cause of Fusarium kernel rot in maize. FB₁ is toxic in domestic and laboratory animals, including pigs. This study investigated the effects of a seven-days-exposure of 1.5mg/kg b.w. FB₁ on the porcine intestinal epithelium. Statistically significant increases in the ratio of sphinganine to sphingosine, as well as alterations of the glycolipid distribution were observed in the jejunum. Using a porcine intestinal epithelial cell line (IPEC-1) derived from jejunum and ileum, we tested the effect of FB₁ in vitro in a time- and dose-dependent fashion. A significant increase in sphinganine concentration was observed after 2 days of FB₁ exposure at concentrations >100μM, or from 6 days of FB₁ exposure at concentration >20μM. We were also able to show that FB₁ exposure at 200μM during 16 days increased the intestinal trans-epithelial flux of FB₁. These data indicate that, in pigs, this mycotoxin acts selectively on jejunum cells as follows: (i) FB₁ affects sphingolipid metabolism, as demonstrated by an increase of the amount of free sphingoid bases in a time- and dose-dependent manner, (ii) a depletion of the glycolipids in plasma membranes is observed, and (iii) an increase occurs in the trans-epithelial flux.

Keywords: Fumonisin B; ₁; Sphingolipids; Glycolipids; Intestinal epithelial cells; Swine; Trans-epithelial flux

Artemisinin effectiveness in erythrocytes is reduced by heme and heme-containing proteins by Napawan Ponmee; Tatsanee Chuchue; Prapon Wilairat; Yongyuth Yuthavong; Sumalee Kamchonwongpaisan (pp. 153-160).

Artemisinin loses its antimalarial activity on prolonged exposure to erythrocytes, especially α-thalassemic erythrocytes. In this report, we show that the major artemisinin-inactivating factor in cytosol of normal erythrocytes was heat-labile but a heat-stable factor from α-thalassemic cells also played a significant role in reducing artemisinin effectiveness, which was shown to be heme released from hemoglobin (Hb). Studies of fractionated lysate from genetically normal erythrocytes revealed that the protein fraction with molecular weight greater than 100kDa was capable of reducing artemisinin effectiveness more readily than lower molecular weight fraction. Catalase and Hb A, but not selenoprotein glutathione peroxidase, were capable of reducing artemisinin effectiveness. Hemin (ferriprotoporphyrin IX) also reduced artemisinin effectiveness in a concentration- and time-dependent manner. It is concluded that heme and heme-containing proteins in erythrocyte are largely responsible for reducing artemisinin effectiveness and may contribute to resistance of Plasmodium falciparum infecting α-thalassemic erythrocytes observed in vitro.

Keywords: Abbreviations; Hb H; α-thalassemia1/α-thalassemia2; Hb H/Hb CS; α-thalassemia1/hemoglobin Constant Spring; Hb A; hemoglobin A; GSH-Px; glutathione peroxidase; IC; ₅₀; 50% inhibitory concentration; MW; molecular weight; kDa; kilodalton; p; p; -valueMalaria; Plasmodium falciparum; Artemisinin; Heme; Catalase; Thalassemia

Interaction and transport characteristics of mycophenolic acid and its glucuronide via human organic anion transporters hOAT1 and hOAT3 by Yuichi Uwai; Hideyuki Motohashi; Yoshie Tsuji; Harumasa Ueo; Toshiya Katsura; Ken-ichi Inui (pp. 161-168).

The immunosuppressant mycophenolate mofetil (MMF) is frequently administered with calcineurin inhibitors and corticosteroids to recipients of organ transplantations. However, the renal handling of the active metabolite mycophenolic acid (MPA) and 7- O-MPA-glucuronide (MPAG) has been unclear. The purpose of the present study was to assess the interaction of MPA and MPAG with the human renal organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8), by conducting uptake experiments using HEK293 cells stably expressing these transporters. MPA and MPAG inhibited the time-dependent uptake of p-[¹⁴C]aminohippurate by hOAT1 and that of [³H]estrone sulfate by hOAT3. The apparent 50% inhibitory concentration (IC₅₀) of MPA for hOAT1 and hOAT3 was estimated at 10.7 and 1.5μM, respectively. In the case of MPAG, the IC₅₀ values were calculated at 512.3μM for hOAT1 and 69.1μM for hOAT3. Eadie–Hofstee plot analyses showed that they inhibited hOAT1 noncompetitively and hOAT3 competitively. No inhibitory effects of tacrolimus, cyclosporin A and azathioprine on transport of p-[¹⁴C]aminohippurate by hOAT1 and of [³H]estrone sulfate by hOAT3 were observed. No transport of MPA by these transporters was observed. On the other hand, the uptake of MPAG into cells was stimulated by the expression of hOAT3, but not hOAT1. These findings propose the possibility that the administration of MMF decreases the renal clearance of drugs which are substrates of hOAT1 and hOAT3. Present data suggest that hOAT3 contributes to the renal tubular secretion of MPAG.

Keywords: Abbreviations; MMF; mycophenolate mofetil; MPA; mycophenolic acid; MPAG; mycophenolic acid glucuronide; OAT; organic anion transporterOrganic anion transporter; Mycophenolic acid; Mycophenolic acid glucuronide; Tubular secretion; Drug interaction

Expression of deoxynucleoside kinases and 5′-nucleotidases in mouse tissues: Implications for mitochondrial toxicity by Svetlana N. Rylova; Saeedeh Mirzaee; Freidoun Albertioni; Staffan Eriksson (pp. 169-175).

Anti-HIV nucleoside therapy can result in mitochondrial toxicity affecting muscles, peripheral nerves, pancreas and adipose tissue. The cytosolic deoxycytidine kinase (dCK; EC 2.7.1.74) and thymidine kinase (TK1; EC 2.7.1.21), the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK; EC 2.7.1.113) as well as 5′-deoxynucleotidases (5′-dNT; EC 3.1.3.5) are enzymes that control rate-limiting steps in formation of intracellular and intra-mitochondrial nucleotides. The mRNA levels and activities of these enzymes were determined in mouse tissues, using real-time PCR and selective enzyme assays. The expression of mRNA for all these enzymes and the mitochondrial deoxynucleotide carrier was detected in all tissues with a 5–10-fold variation. TK1 activities were only clearly detected in spleen and testis, while TK2, dGK and dCK activities were found in all tissues. dGK activities were higher than any other dNK in all tissues, except spleen and testis. In skeletal muscle dGK activity was 5-fold lower, TK2 and dCK levels were 10-fold lower as compared with other tissues. The variation in 5′-dNT activities was about eight-fold with the highest levels in brain and lowest in brown fat. Thus, the salvage of deoxynucleosides in muscles is 5–10-fold lower as compared to other non-proliferating tissues and 100-fold lower compared to spleen. These results may help to explain tissue specific toxicity observed with nucleoside analogs used in HIV treatment as well as symptoms in inherited mitochondrial TK2 deficiencies.

Keywords: Abbreviations; AMV-RT; avian myeloblastosis virus reverse transcriptase; cN-1b; cytosolic 5′-nucleotidase 1b; cN-2; cytosolic 5′-nucleotidase 2; dCK; deoxycytidine kinase; dGK; deoxyguanosine kinase; DNC; deoxynucleotide carrier; dNK; deoxynucleoside kinase; dNT-1; deoxynucleotidase 1; ecto-NT; ecto 5′-nucleotidase; GAPDH; glyceraldehyde-3-phosphate-dehydrogenase; HIV; human immunodeficiency virus; mt; mitochondria; NRTI; nucleoside reverse transcriptase inhibitor; NRTI-TP; NRTI triphosphate; 5′-dNT; 5′-nucleotidases; TK1; thymidine kinase 1; TK2; thymidine kinase 2Nucleoside kinases; Nucleotidases; Chemotherapy; Metabolism; Mitochondria; Toxicity

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Biochemical Pharmacology (v.74, #1)

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Biochemical Pharmacology (v.74, #1)