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Biochemical Pharmacology (v.74, #10)

Editorial Advisory Board (pp. iii).

Relevance of extracellular and intracellular interactions of camptothecins as determinants of antitumor activity by Giovanni Luca Beretta; Franco Zunino (pp. 1437-1444).

Camptothecins are potent antitumor agents that stabilize the covalent binding of topoisomerase I to DNA forming a reversible ternary complex which, following collision with the replication forks, converts the single-strand breaks into lethal double-strand breaks. This cytotoxic mechanism has been originally ascribed to the closed lactone form, because opening of the lactone ring resulted in loss of antitumor activity. Since the lipophilic lactone favours passive diffusion into the cancer cells, the stability of the closed form is expected to be predictive for activity. Thus, the in vivo pharmacological behavior of camptothecins, which is dependent on the pH-dependent dynamics, is likely a critical determinant of their antitumor efficacy and therapeutic index. The physicochemical properties could influence a number of cellular and in vivo interactions, including stability of the ternary DNA–enzyme–drug complex, binding to serum proteins, recognition by transport systems. These interactions are also implicated in the processes responsible of toxic side effects and drug resistance which are major limitations of the efficacy of camptothecin-based therapy. A number of strategies have been developed to overcome the limitations associated with the peculiar in vivo reactivity and the reversibility of drug–target interaction. Modifications with hydrophilic or lipophilic substituents at specific positions may have a variable (and somewhat opposite) influence on interaction with the intracellular target and plasma proteins and on recognition by membrane transporters. Here, we highlight the interactions of camptothecins which could be exploited to optimize therapeutic efficacy.

Keywords: Camptothecin; Antitumor pharmacology; DNA damage; Transporters; Human serum albumin; DNA topoisomerase

Deglycosylated bleomycin induces apoptosis in lymphoma cell via c-jun NH₂-terminal kinase but not reactive oxygen species by Souhir Brahim; Laurent Prévotat; Sonia Yatouji; Delphine Mérino; Marion Cortier; Cédric Rébé; Olivier Micheau; Abderraouf Kenani; Ali Bettaieb (pp. 1445-1455).

Bleomycin (BLM) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of lung fibrosis. This side effect is related to the ability of the drug to generate reactive oxygen species in lung cells. In the present study, we evaluated the consequences of deglycosylation of BLM in term of cytotoxic activity and generation of reactive oxygen species. When tested on U937 human lymphoma cells, both compounds generated a typical apoptotic phenotype. Cell death induction was associated with Bax oligomerization, dissipation of the mitochondrial membrane potential, release of cytochrome c, caspase activation, chromatin condensation and internucleosomal degradation. Whereas both reactive oxygen species and c-jun NH₂-terminal kinase (JNK) inhibitors prevented BLM-induced U937 cell death, only JNK inhibition prevented deglycosylated BLM-mediated cell death. Both compounds induced clustering of TRAIL receptors (DR4 and DR5) and Fas at the cell surface but neither a chimeric soluble DR5 receptor that inhibits TRAIL-induced cell death nor a dominant negative version of the adaptor molecule Fas-associated death domain prevented BLM-induced cytotoxicity. These observations indicate that deglycosylation of BLM does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway but prevents induction of reactive oxygen species. This observation suggests that deglycosylated BLM could exhibit less toxic side effects and could warrant its use in clinic.

Keywords: Bleomycin; Lymphoma; Apoptosis; ROS; JNK; Death receptors

Lipid rafts and metabolic energy differentially determine uptake of anti-cancer alkylphospholipids in lymphoma versus carcinoma cells by Stefan R. Vink; Arnold H. van der Luit; Jeffrey B. Klarenbeek; Marcel Verheij; Wim J. van Blitterswijk (pp. 1456-1465).

Perifosine is a member of the class of synthetic alkylphospholipids (APLs) and is being evaluated as anti-cancer agent in several clinical trials. These single-chain APLs accumulate in cellular membranes and disturb lipid-dependent signal transduction, ultimately causing apoptosis in a variety of tumor cells. The APL prototype edelfosine was previously found to be endocytosed by S49 mouse lymphoma cells via lipid rafts. An edelfosine-resistant cell variant, S49^AR, was found to be cross-resistant to other APLs, including perifosine. This resistance was due to defective synthesis of the raft constituent sphingomyelin, which abrogated APL cellular uptake. Sensitivity of S49 cells to edelfosine was higher than perifosine, which correlated with a relatively higher uptake. Human KB epidermal carcinoma cells were much more sensitive to APLs than S49 cells. Their much higher APL uptake was highly dependent on intracellular ATP and ambient temperature, and was blocked by chlorpromazine, independent of canonical endocytic pathways. We found no prominent role of lipid rafts for APL uptake in these KB cells; contrary to S49^AR cells, perifosine-resistant KBr cells display normal sphingomyelin synthesis, whereas APL uptake by the responsive KB cells was insensitive to treatment with methyl-β-cyclodextrin, a cholesterol-sequestrator and inhibitor of raft-mediated endocytosis. In conclusion, different mechanisms determine APL uptake and consequent apoptotic toxicity in lymphoma versus carcinoma cells. In the latter cells, APL uptake is mainly determined by a raft- and endocytosis-independent process, but metabolic energy-dependent process, possibly by a lipid transporter.

Keywords: Abbreviations; APL; alkylphospholipid; MβCD; methyl-β-cyclodextrin; PC; phosphatidylcholine; ErPC; erucylphosphocholine; SM; sphingomyelin; PBS; phosphate-buffered saline; TLC; thin-layer chromatographyAlkylphospholipids; Perifosine; Edelfosine; Endocytosis; Lipid rafts; Plasma membrane traversal; Resistance

Effects of nonpeptide vasopressin V₂ antagonist tolvaptan in rats with heart failure by Punniyakoti T. Veeraveedu; Kenichi Watanabe; Meilei Ma; Suresh S. Palaniyandi; Ken’ichi Yamaguchi; Kenji Suzuki; Makoto Kodama; Yoshifusa Aizawa (pp. 1466-1475).

Similar to other neurohormones that are activated in chronic heart failure (CHF), circulating arginine vasopressin (AVP) is elevated in patients with CHF. The precise role of AVP in the pathophysiology of cardiovascular disease is controversial. AVP is a peptide hormone that contributes to water retention and vasoconstriction in CHF through effects on V₂ and V_1a receptors, respectively. In the present study, the effect of V₂ receptor (V₂R) blockade using tolvaptan was assessed in a rat model of myosin-induced experimental autoimmune myocarditis. CHF was elicited in Lewis rats by immunization with porcine cardiac myosin, and 28 days after immunization rats were treated for 28 days with oral tolvaptan (3 or 10mg/(kgday)) or vehicle. CHF was characterized by left ventricular remodeling and impaired systolic and diastolic function. Chronic V₂R blockade increased urine volume and urinary AVP excretion and decreased urine osmolality but had no natriuretic effect, and as a result caused increases in plasma osmolality and sodium. High doses of tolvaptan markedly elevated electrolyte-free water clearance. V₂R blockade did not activate the renin–angiotensin system, not influence cardiac remodeling, cardiac function, or survival. The upregulation of aquaporin 2 protein in the kidney of CHF rats was inhibited by the administration of V₂R antagonist. These results suggest that in a rat model of CHF, AVP plays a major role in water retention through the renal V₂R.

Keywords: Vasopressin; Vasopressin receptor antagonists; Hyponatremia; Water excretion; Chronic heart failure; Aquaporin 2

Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone by Chellappagounder Thangavel; Ravindra N. Dhir; Denys V. Volgin; Bernard H. Shapiro (pp. 1476-1484).

CYP2C11, the most commonly expressed isoform of cytochrome P450 in male rat liver, was measured in spleen, thymus and bone marrow by quantitative real-time PCR and enhanced Western blotting. CYP2C11 concentrations in the lymphoid tissues were a fraction of that observed in liver, but like the liver, were sexually dimorphic (M>F) with mRNA and protein levels in agreement. Although the response to hypophysectomy varied according to tissue and sex, expression levels of CYP2C11 in all measured tissues remained greater in males. Further differences in CYP2C11 expression between liver and lymphoid tissue were observed following restoration of the circulating masculine growth hormone profile in hypophysectomized rats. In contrast to the liver where the renaturalized growth hormone profile elevated CYP2C11 expression in both sexes, the response was opposite in spleen and thymus with isoform concentrations declining in both sexes. Lastly, the divergent response of CYP2C11 between the liver and immune system was examined in cultured splenocytes exposed to different mitogens. In contrast to the dramatic depletion of CYP2C11 reported in proliferating hepatocytes, mitogen-stimulation resulted in a significant elevation in splenocyte CYP2C11 expression. In summary, we report for the first time that thymus, spleen and bone marrow express, albeit nominal, sex-dependent levels of CYP2C11 (M>F) whose regulation appears to be under some hormonal control, but very different from that of the hepatic isoform.

Keywords: Spleen, Cytochrome P450; Thymus, Cytochrome P450; Bone marrow, Cytochrome P450; CYP2C11, Spleen, Thymus, Bone marrow; Lymphoid CYP2C11, Growth hormone regulation; Splenic CYP2C11, Mitogen effects

NSAIDs enhance proteasomic degradation of survivin, a mechanism of gastric epithelial cell injury and apoptosis by S.-K. Chiou; S. Mandayam (pp. 1485-1495).

NSAIDs cause severe gastrointestinal injury, in part by suppressing survivin, an inhibitor of apoptosis protein, both in cultured gastric epithelial cells and in human and rat gastric mucosa. The mechanism(s) of survivin down-regulation by NSAIDs is unclear. In this study, we examined whether NSAID treatment decreases survivin mRNA expression and/or enhances degradation of survivin protein via ubiquitin proteasome system in rat gastric mucosal, RGM-1 cells, and whether survivin overexpression prevents indomethacin-induced cell injury and apoptosis. Effects of indomethacin on survivin mRNA expression, survivin protein half-life and ubiquitination were examined in RGM-1 cells. Proteasome inhibitors were utilized to prevent indomethacin-induced survivin protein degradation in RGM-1 cells. The effects of stable overexpression of survivin on indomethacin-induced RGM-1 cell injury and apoptosis were examined. Results showed: (1) Indomethacin treatment did not alter survivin mRNA expression, but significantly reduced survivin protein half-life from 1.5h to approximately 1h and increased survivin ubiquitination. (2) Inhibition of ubiquitin proteasome prolonged survivin protein half-life to over 2h and inhibited indomethacin-induced survivin degradation. (3) Overexpression of survivin significantly reduced indomethacin-induced cell injury and apoptosis. In conclusion, indomethacin treatment enhances degradation of survivin via the ubiquitin proteasome machinery in RGM-1 cells, and maintenance of survivin levels is important for prevention of gastric epithelial cell injury and apoptosis.

Keywords: Apoptosis; NSAIDs; Survivin; Gastrointestinal cell injury; Ubiquitin proteasome; Protein degradation

The intestinal anti-inflammatory effects of the novel agent UR-1505 in the TNBS model of rat colitis are mediated by T-lymphocyte inhibition by Elvira Bailón; Desirée Camuesco; Ana Nieto; Angel Concha; Alberto Fernández de Arriba; Juan Román; Isabel Ramis; Manuel Merlos; Antonio Zarzuelo; Julio Gálvez; Mònica Comalada (pp. 1496-1506).

UR-1505 is a novel pentafluoropropoxy derivative of salicylic acid, selected from a series of salicylate derivatives, according to their activity as inhibitors of T-lymphocyte activation. This study describes the anti-inflammatory activity of UR-1505 on trinitrobenzenesulphonic acid-induced colitis in rat, an experimental model that resembles to Crohn's disease (CD), as well as its in vitro effects on T-cells and bone marrow-derived macrophages (BMDM) activation. UR-1505 showed intestinal anti-inflammatory effect, associated with reduced colonic levels of TNFα and LTB₄, inhibition of the expression of IFNγ and iNOS, and lower colonic leukocyte infiltration. The in vitro assays revealed that UR-1505 also inhibited T-lymphocyte proliferation and IL-12/IFNγ production, two of the main pro-inflammatory cytokines involved in the pathogenesis of CD. However, UR-1505 did not modify LPS- nor IFNγ-induced activation in BMDM. Thus, UR-1505 specifically affects T-cells without modifying the activation of BMDM. In conclusion, the intestinal anti-inflammatory activity of UR-1505 seems to be mediated by a reduction in the recruitment of immune cells to the inflammatory foci, together with the inhibition of T-cell activation. These results suggest that UR-1505 may be an interesting candidate to be explored for the treatment of CD.

Keywords: Abbreviations; BMDM; bone marrow-derived macrophage; CsA; cyclosporine A; Con A; concanavalin A; COX; cyclooxygenase; CD; Crohn's disease; GSH; glutathione; IBD; inflammatory bowel disease; iNOS; inducible nitric oxide synthase; LTB; ₄; leukotriene B; ₄; MPO; myeloperoxidase; SA; salicylic acid; TNBS; trinitrobenzenesulphonic acid; UC; ulcerative colitisSalicylates; Cyclosporine A; Th1 inflammation; Crohn's disease; Lymphocyte; Inflammatory bowel disease

Phosphodiesterase isozymes involved in regulation of HCO₃⁻ secretion in isolated mouse duodenum in vitro by Msamune Hayashi; Kazutomo Kita; Yumi Ohashi; Eitaro Aihara; Koji Takeuchi (pp. 1507-1513).

We examined the effects of various isozyme-selective PDE inhibitors on HCO₃⁻ secretion in the mouse duodenum in vitro and investigated which type(s) of phosphodiesterase (PDE) isozymes are involved in the response to PGE₂ and NO. The duodenal mucosa of male DDY mice was stripped of the muscle layer and mounted on an Ussing chamber, and HCO₃⁻ secretion was measured at pH 7.0 by a pH-stat method using 2mM HCl. Both PGE₂ and NOR-3 (NO donor) increased HCO₃⁻ secretion in the mouse duodenum in vitro, and the response to PGE₂ was inhibited by both EP3 and EP4 antagonists but not EP1 antagonist, while that to NOR-3 was inhibited by methylene blue. IBMX, a nonselective PDE inhibitor, significantly increased basal HCO₃⁻ secretion and potentiated the responses to both PGE₂ and NOR-3. Likewise, vinpocetine (PDE1 inhibitor) and cilostamide (PDE3 inhibitor) also increased the basal secretion at high doses and potentiated the HCO₃⁻ response to PGE₂ at doses that had no effect by themselves on the basal secretion. By contrast, the HCO₃⁻ stimulatory action of NOR-3 was significantly potentiated by vinpocetine but not cilostamide. Inhibitors of other PDE subtypes had no effect on the HCO₃⁻ secretion under basal or stimulated conditions. Both PDE1 and PDE3 mRNAs were expressed in the duodenal mucosa. These results suggested that PDE1 and PDE3 are involved in the regulation of duodenal HCO₃⁻ secretion and that the response to PGE₂ is associated with both PDE1 and PDE3, while the response to NO is mainly modulated by PDE1.

Keywords: Duodenal HCO; ₃; ⁻; secretion; Prostaglandin E; ₂; Nitric oxide; Phosphodiesterase isozyme; cGMP; cAMP

Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions by Timo Burster; Viviana Marin-Esteban; Bernhard O. Boehm; Shannon Dunn; Olaf Rotzschke; Kirsten Falk; Ekkehard Weber; Steven H.L. Verhelst; Hubert Kalbacher; Christoph Driessen (pp. 1514-1523).

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

Keywords: Abbreviations; AEP; asparagine-specific endopeptidase; APC; antigen presenting cells; APL; altered peptide ligand; BLC; B-lymphoblastoid cell; Cat; cathepsin; EAE; experimental allergic encephalomyelitis; HPSEC; high performance size exclusion chromatography; HRP; horseradish-peroxidase; HLA; human leukocyte antigen; Ii; MHC class II invariant chain; MBP; myelin basic protein; MS; multiple sclerosisAntigen presentation/processing; Antigens/peptides/epitopes; Autoimmunity; MHC

Inhibition of human and mouse plasma membrane bound NTPDases by P2 receptor antagonists by Mercedes N. Munkonda; Gilles Kauffenstein; Filip Kukulski; Sébastien A. Lévesque; Charlène Legendre; Julie Pelletier; Élise G. Lavoie; Joanna Lecka; Jean Sévigny (pp. 1524-1534).

The plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase)-1, 2, 3 and 8 are major ectonucleotidases that modulate P2 receptor signaling by controlling nucleotides’ concentrations at the cell surface. In this report, we systematically evaluated the effect of the commonly used P2 receptor antagonists reactive blue 2, suramin, NF279, NF449 and MRS2179, on recombinant human and mouse NTPDase1, 2, 3 and 8. Enzymatic reactions were performed in a Tris/calcium buffer, commonly used to evaluate NTPDase activity, and in a more physiological Ringer modified buffer. Although there were some minor variations, there were no major changes either in the enzymatic activity or in the profile of NTPDase inhibition between the two buffers. Except for MRS2179, all other antagonists significantly inhibited these ecto-ATPases; NTPDase3 being the most sensitive to inhibition and NTPDase8 the most resistant. Estimated IC₅₀ showed that human NTPDases were generally more sensitive to the P2 receptor antagonists tested than the corresponding mouse isoforms. NF279 and reactive blue 2 were the most potent inhibitors of NTPDases which almost completely abrogated their activity at the concentration of 100μM. In conclusion, reactive blue 2, suramin, NF279 and NF449, at the concentrations commonly used to antagonize P2 receptors, inhibit the four major ecto-ATPases. This information may reveal useful for the interpretation of some pharmacological studies of P2 receptors. In addition, NF279 is a most potent non-selective NTPDase inhibitor. Although P2 receptor antagonists do not display a strict selectivity toward NTPDases, their IC₅₀ values may help to discriminate some of these enzymes.

Keywords: Abbreviations; MRS2179; 2′-Deoxy-; N; ⁶; -methyladenosine 3′,5′-bisphosphate; NF279; 8,8′-[Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid; NF449; 4,4′,4″,4‴-[Carbonylbis(imino-5,1,3-benzenetriyl-; bis; (carbonylimino))]; tetrakis; -1,3-benzenedisulfonic acid; NTPDase; nucleoside triphosphate diphosphohydrolaseATP; ADP; Ectonucleotidase; Ecto-ATPase; NTPDase; P2 receptor; Reactive blue 2; Suramin; NF279; NF449; MRS2179; Antagonist; Inhibitor

Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists by Zofia Duniec-Dmuchowski; Ewa Ellis; Stephen C. Strom; Thomas A. Kocarek (pp. 1535-1540).

The liver X receptor (LXR) agonists, 24( S),25-epoxycholesterol and T0901317, were previously shown to be capable of inducing CYP3A expression in primary cultured rodent hepatocytes through activation of the pregnane X receptor (PXR). In this study, the abilities of these two LXR agonists to regulate CYP3A4 and CYP2B6 mRNA expression in primary cultures of human hepatocytes were evaluated. Treatment with 10 or 30μM of the endogenous oxysterol, 24( S),25-epoxycholesterol, had no effect on CYP3A4 mRNA content in five preparations of primary cultured human hepatocytes, while 30μM 24( S),25-epoxycholesterol treatment increased CYP2B6 mRNA content by approximately two-fold. By comparison, treatment with the synthetic LXR agonist, T0901317, potently increased CYP3A4 and CYP2B6 mRNA levels in the human hepatocyte cultures, producing multi-fold increases at 10nM. Using a HepG2-based transactivation assay, T0901317 activated human PXR with an EC₅₀∼20nM, which was more than 10-fold lower than that of the potent PXR ligand, SR-12813, while treatment with 24( S),25-epoxycholesterol failed to induce reporter expression in this assay. Therefore, while 24( S),25-epoxycholesterol-mediated PXR activation and CYP3A induction does not appear to be conserved from rodent to human, T0901317 is among the most potent known activators of human PXR.

Keywords: Liver X receptor agonists; Pregnane X receptor; T0901317; CYP3A4; CYP2B6; Human hepatocytes

Nuclear translocation of telomerase reverse transcriptase and calcium signaling in repression of telomerase activity in human lung cancer cells by fungal immunomodulatory protein from Ganoderma tsugae by Chien-Huang Liao; Yi-Min Hsiao; Gwo-Tarng Sheu; Jinghua Tsai Chang; Po-Hui Wang; Ming-Fang Wu; Gow-Jen Shieh; Chung-Ping Hsu; Jiunn-Liang Ko (pp. 1541-1554).

Recombinant fungal immunomodulatory protein, reFIP-gts, was cloned from Ganoderma tsugae and purified. In our previous study, it was shown that reFIP-gts has anti-telomerase effects in A549 cells. Here, we proved that reFIP-gts entry into the cell and localization in endoplasmic reticulum can result in ER stress, thereby increasing ER stress markers (CHOP/GADD153) and intracellular calcium release in A549 cells. The use of calcium chelator restores reFIP-gts-mediated reduction in telomerase activity. These results strongly suggest that ER stress induces intracellular calcium release and results in inhibition of telomerase activity. Although reFIP-gts decreased hTERT mRNA level in both A549 and H1299 cells, only the telomerase activity in A549 cells was inhibited. Surprisingly, we found that reFIP-gts induces translocation of hTERT from the nucleus into the cytosol in A549 cells but not in H1299 cells. Using leptomycin B, nuclear export inhibitor, we showed that hTERT is not transported. Using MG132, a proteasome inhibitor, reFIP-gts also prevents hTERT translocation from proteasome degradation. Taken together, these results indicate that reFIP-gts inhibits telomerase activity in lung cancer cells through nuclear export mechanisms, which might be mediated by ER stress-induced intracellular calcium level.

Keywords: Abbreviations; reFIP-gts; recombinant fungal immunomodulatory protein; Ganoderma tsugae; hTERT; human telomerase reverse transcriptase; ER; endoplasmic reticulum; GADD153; growth arrest- and DNA damage-inducible gene 153; TG; thapsigarginFungal immunomodulatory protein; ER stress; hTERT; Nuclear export

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Biochemical Pharmacology (v.74, #10)