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Biochemical Pharmacology (v.82, #2)

Editorial Advisory Board (pp. iii).

Recent advances in the development of coagulation factors and procoagulants for the treatment of hemophilia by Robert G. Schaub (pp. 91-98).

Hemophilia is a family of rare bleeding disorders. The two primary types, hemophilia A and hemophilia B, are caused by recessive X-chromosome linked mutations that result in deficiency of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Clinically, hemophilia is manifested by spontaneous bleeding, particularly into the joints (haemarthrosis) and soft tissue, and excessive bleeding following trauma or surgery. The total overall number of hemophilia patients worldwide is approximately 400,000, however only about 100,000 of these individuals are treated. The first treatment of hemophilia was initiated when it was determined that the clotting deficiency could be corrected by a plasma fraction taken from normal blood. The discovery of factor VIII enrichment by cryoprecipitation of plasma opened a new era of therapy which eventually led to the production of factor concentrates and the subsequent development of highly purified forms of plasma factors. The most significant improvements have been the availability of recombinant forms of factors VIII and IX. Unfortunately, recombinant factors still retain some of the limitations of plasma concentrates. These limitations include development of antibody responses in patients and the relatively short half-life of the molecules requiring frequent injection to maintain effective concentration. Treatment beyond replacement of native factors has been tried. They include the development of modified factor VIII and IX molecules with improved potency, stability and circulating half-life and enhancement of a prothrombotic responses and/or stabilization of coagulation factors via inhibition of key negative regulatory pathways. These approaches will be reviewed in this commentary.

Keywords: Hemophilia; Factor IX; Factor VIII; Coagulation

The clinical potential of the acyclic (and cyclic) nucleoside phosphonates. The magic of the phosphonate bond by Erik De Clercq (pp. 99-109).

The use of the acyclic nucleoside phosphonates, starting with ( S)-HPMPA as the prototype, yielded three clinically approved antiviral drugs, cidofovir for the treatment of CMV retinitis in AIDS patients, adefovir dipivoxil for the treatment of chronic hepatitis B and tenofovir disoproxil fumarate for the treatment of HIV infections (AIDS) and HBV infections. This era has now grown to many more acyclic (and cyclic) nucleoside phosphonates (such as the “open ring” DAPy and Fd4A phosphonates) and alkoxyalkyl and phosphonoamidate prodrugs thereof, as well as new clinical applications, including new drug combination regimens for the treatment of AIDS, the chemoprophylaxis of HIV infections, and the anticancer potential against some malignant disorders.

Keywords: Antiviral; Phosphonate; Nucleotide

Second generation benzofuranone ring substituted noscapine analogs: Synthesis and biological evaluation by Ram Chandra Mishra; Prasanthi Karna; Sushma Reddy Gundala; Vaishali Pannu; Richard A. Stanton; Kamlesh Kumar Gupta; M. Hope Robinson; Manu Lopus; Leslie Wilson; Maged Henary; Ritu Aneja (pp. 110-121).

Computer models depicting noscapine within the colchicine-binding domain of tubulin suggest that increasing substitution group size at position-7 of the benzofuranone ring will increase steric strain and decrease binding affinity.Microtubules, composed of α/β tubulin heterodimers, represent a validated target for cancer chemotherapy. Thus, tubulin- and microtubule-binding antimitotic drugs such as taxanes and vincas are widely employed for the chemotherapeutic management of various malignancies. Although quite successful in the clinic, these drugs are associated with severe toxicity and drug resistance problems. Noscapinoids represent an emerging class of microtubule-modulating anticancer agents based upon the parent molecule noscapine, a naturally occurring non-toxic cough-suppressant opium alkaloid. Here we report in silico molecular modeling, chemical synthesis and biological evaluation of novel analogs derived by modification at position-7 of the benzofuranone ring system of noscapine. The synthesized analogs were evaluated for their tubulin polymerization activity and their biological activity was examined by their antiproliferative potential using representative cancer cell lines from varying tissue-origin [A549 (lung), CEM (lymphoma), MIA PaCa-2 (pancreatic), MCF-7 (breast) and PC-3 (prostate)]. Cell-cycle studies were performed to explore their ability to halt the cell-cycle and induce subsequent apoptosis. The varying biological activity of these analogs that differ in the nature and bulk of substituent at position-7 was rationalized utilizing predictive in silico molecular modeling.

Keywords: Noscapine; Anticancer activity; Tubulin-binding; Cell cycle

The turnover of estrogen receptor α by the selective estrogen receptor degrader (SERD) fulvestrant is a saturable process that is not required for antagonist efficacy by Suzanne E. Wardell; Jeffrey R. Marks; Donald P. McDonnell (pp. 122-130).

It has become apparent of late that even in tamoxifen and/or aromatase resistant breast cancers, ERα remains a bona fide therapeutic target. Not surprisingly, therefore, there has been considerable interest in developing Selective ER Degraders (SERDs), compounds that target the receptor for degradation. Currently, ICI 182,780 (ICI, fulvestrant) is the only SERD approved for the treatment of breast cancer. However, the poor pharmaceutical properties of this injectable drug and its lack of superiority over second line aromatase inhibitors in late stage breast cancer have negatively impacted its clinical use. These findings have provided the impetus to develop second generation, orally bioavailable SERDs with which quantitative turnover of ERα in tumors can be achieved. Interestingly however, the contribution of SERD activity to fulvestrant efficacy is unclear, making it difficult to define the characteristics desired of the next generation of ER antagonists. It is of significance therefore, that we have determined that the antagonist activity of ICI and its ability to induce ERα degradation are not coupled processes. Specifically, our results indicate that it is the ability of ICI to interact with ERα and to (a) competitively displace estradiol and (b) induce a conformational change in ER incompatible with transcriptional activation that are likely to be the most important pharmacological characteristics of this drug. Collectively, these data argue for a renewed emphasis on the development of high affinity, orally bioavailable pure antagonists and suggest that SERD activity though proven effective may not be required for ERα antagonism in breast cancer.

Keywords: Estrogen receptor; Degradation; ICI 182,780; Fulvestrant; SERD

Point mutations in human guanylate kinase account for acquired resistance to anticancer nucleotide analogue PMEG by Helena Mertlíková-Kaiserová; Michaela Rumlová; Eva Tloušťová; Eliška Procházková; Antonín Holý; Ivan Votruba (pp. 131-138).

Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S³⁵N V¹⁶⁸F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.

Keywords: Abbreviations; PMEG; 9-[2-(phosphonomethoxyethyl)guanine]; PMEDAP; 9-[2-(phosphonomethoxyethyl)diaminopurine]; PMEA; 9-[2-(phosphonomethoxyethyl)adenine]; GUK; guanylate kinase; AK; adenylate kinase; GMP; guanosine monophosphate; AMP; adenosine monophosphate; ANP; acyclic nucleoside phosphonate; CdA; 2-chloro-2′-deoxyadenosine (cladribine); FUDR; 5-fluoro-2′-deoxyuridineResistance; Acyclic nucleoside phosphonates; PMEG; PMEDAP; Guanylate kinase; Adenylate kinase

Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment by Joachim Gullbo; Mårten Fryknäs; Linda Rickardson; Padraig Darcy; Maria Hägg; Malin Wickström; Sadia Hassan; Gunnar Westman; Slavica Brnjic; Peter Nygren; Stig Linder; Rolf Larsson (pp. 139-147).

Screening primary cultures of patient tumor cells in response to chemically diverse library of compounds identified VLX 50 as an iron chelator with tumor selective activity.Primary cultures of patient tumor cells (PCPTC) have been used for prediction of diagnosis-specific activity and individual patient response to anticancer drugs, but have not been utilized as a model for identification of novel drugs in high throughput screening. In the present study, ovarian carcinoma cells from three patients were tested in response to a library of 3000 chemically diverse compounds. Eight hits were retrieved after counter screening using normal epithelial cells, and one of the two structurally related hit compounds was selected for further preclinical evaluation. This compound, designated VLX 50, demonstrated a broad spectrum of activity when tested in a panel of PCPTCs representing different forms of leukemia and solid tumors and displayed a high tumor to normal cell activity. VLX 50 induced delayed cell death with some features of classical apoptosis. Significant in vivo activity was confirmed on primary cultures of human ovarian carcinoma cells in mice using the hollow fiber model. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. This query signature was analyzed using the Gene Set Enrichment Analysis and the Connectivity Map database. Strong connections to hypoxia inducible factor 1 and iron chelators were retrieved. The mechanistic hypothesis of intracellular iron depletion leading to hypoxia signaling was confirmed by a series of experiments. The results indicate the feasibility of using PCPTC for cancer drug screening and that intracellular iron depletion could be a potentially important strategy for cancer therapy.

Keywords: Abbreviations; ALL; acute lymphocytic leukemia; AML; acute myelocytic leukemia; CLL; chronic lymphocytic leukemia; CML; chronic myelocytic leukemia; NHL; non-Hodgkin's lymphoma; PBMC; peripheral blood mononuclear cellsScreening; Ovarian cancer; Primary cultures; Iron depletion

Ginsenoside Rb1 prevents homocysteine-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition by Tao-Hua Lan; Zhi-Wei Xu; Zhi Wang; Yi-Ling Wu; Wei-Kang Wu; Hong-Mei Tan (pp. 148-155).

Hyperhomocysteinemia (HHcy), a risk factor for cardiovascular disease, is associated with endothelial dysfunction. Ginsenoside Rb1, the major active constituent of ginseng, potently attenuates homocysteine (Hcy)-induced endothelial damage. However, the underlying mechanism remains unknown. In this study, we have investigated the effect of Ginsenoside Rb1 on Hcy-induced endothelial dysfunction and its underlying signal pathway in vivo and in vitro. Ginsenosides prevented Hcy-induced impairment of endothelium-dependent relaxation and Rb1 reversed Hcy-induced reduction of NO production in a dose-dependent manner as detected by nitrate reductase method. Rb1 activated serine-1177 phosphorylation of endothelial nitric oxide synthase (eNOS) and serine-473 phosphorylation of Akt, while inhibited threonine-495 phosphorylation of eNOS as detected by western blotting. Rb1-induced phosphorylation of serine-1177 was significantly inhibited by wortmannin, PI3K inhibitor or SH-5, an Akt inhibitor, and partially reversed by Phorbol 12-myristate 13-acetate (PMA), a PKC activator. PMA also stimulated phosphorylation of threonine-495 which was inhibited by Rb1. Here we show for the first time that Rb1 prevents Hcy-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.

Keywords: Abbreviations; Akt; protein kinase B; ECGS; endothelial cell growth supplement; eNOS; endothelial nitric oxide synthase; FBS; fetal bovine serum; GS; ginsenosides; Hcy; homocysteine; HHcy; Hyperhomocysteinemia; HUVECs; human umbilical vein endothelial cells; l; -NAME; N-nitro-; l; -arginine methyl ester hydrochloride; M199; medium 199; NO; nitric oxide; PBS; phosphate-buffered saline; PI3K; phosphoinositide 3-kinase; PKC; protein kinase C; PMA; Phorbol 12-myristate 13-acetate; SNP; sodium nitroprusside; SST; Succinylsulfathiazole; vWF; von Willebrand factorGinsenoside Rb1; Homocysteine; NO; PI3K/Akt; PKC

Tumor necrosis factor alpha-mediated inhibition of erythropoiesis involves GATA-1/GATA-2 balance impairment and PU.1 over-expression by Christine Grigorakaki; Franck Morceau; Sébastien Chateauvieux; Mario Dicato; Marc Diederich (pp. 156-166).

Many physiological perturbations can cause anemia. In cancer patients, activation of the immune system leads to the production of proinflammatory cytokines including tumor necrosis factor alpha (TNFα), that have been shown to inhibit red-cell production via poorly understood mechanisms. Treatment of anemia by human recombinant erythropoietin (EPO) is strongly suspected to induce tumor growth.This study focuses on the mechanisms involved in TNFα-mediated inhibition of erythropoiesis. CD34⁺ hematopoietic stem/progenitor cells (HSPCs) were isolated from human cord blood. Erythropoiesis was achieved in vitro by stimulating cells with EPO. We show that TNFα clearly affected erythroid development, as assessed by May-Grünwald/Giemsa staining, flow cytometry analysis and fluorescent microscopy. The amount of hemoglobin-producing cells as well as the expression of GATA-1 target erythro-specific genes (EPO receptor, glycophorin A and globins) was found decreased after TNFα treatment of HSPC. In correlation, TNFα induced the expression of the transcription factors GATA-2 and PU.1, described as inhibitors of erythropoiesis. In this regard, TNFα promoted the formation of the GATA-1/PU.1 complex that has been reported to block the transcriptional activity of GATA-1. Our results clearly demonstrate that TNFα prevents EPO-mediated erythropoiesis of HSPC as an early event, by directly affecting erythroid cell development.

Keywords: Anemia; Inflammation; TNFα; GATA-1; GATA-2; PU.1

Cyanidin-3-glucoside suppresses B[a]PDE-induced cyclooxygenase-2 expression by directly inhibiting Fyn kinase activity by Tae-Gyu Lim; Jung Yeon Kwon; Jiyoung Kim; Nu Ry Song; Kyung Mi Lee; Yong-Seok Heo; Hyong Joo Lee; Ki Won Lee (pp. 167-174).

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) is a well-known carcinogen that is associated with skin cancer. Abnormal expression of cyclooxygenase-2 (COX-2) is an important mediator in inflammation and tumor promotion. We investigated the inhibitory effect of cyanidin-3-glucoside (C3G), an anthocyanin present in fruits, on B[a]PDE-induced COX-2 expression in mouse epidermal JB6 P+ cells. Pretreatment with C3G resulted in the reduction of B[a]PDE-induced expression of COX-2 and COX-2 promoter activity. The activation of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) induced by B[a]PDE was also attenuated by C3G. C3G attenuated the B[a]PDE-induced phosphorylation of MEK, MKK4, Akt, and mitogen-activated protein kinases (MAPKs), but no effect on the phosphorylation of the upstream MAPK regulator Fyn. However, kinase assays demonstrated that C3G suppressed Fyn kinase activity and C3G directly binds Fyn kinase noncompetitively with ATP. By using PP2, a pharmacological inhibitor for SFKs, we showed that Fyn kinase regulates B[a]PDE-induced COX-2 expression by activating MAPKs, AP-1 and NF-κB. These results suggest that C3G suppresses B[a]PDE-induced COX-2 expression mainly by blocking the activation of the Fyn signaling pathway, which may contribute to its chemopreventive potential.

Keywords: Cyanidin-3-glucoside; Benzo[a]pyrene-7,8-diol-9,10-epoxide; Cyclooxygenase-2; Fyn; Chemoprevention

Mouse γ-butyrobetaine dioxygenase is regulated by peroxisome proliferator-activated receptor α through a PPRE located in the proximal promoter by Gaiping Wen; Hagen Kühne; Christine Rauer; Robert Ringseis; Klaus Eder (pp. 175-183).

Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5′-truncation BBD promoter reporter constructs revealed that one PPRE located at −75 to −87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.

Keywords: Carnitine; γ-Butyrobetaine dioxygenase; Peroxisome proliferator-activated receptor α; Mouse

The tyrosine kinase inhibitor sorafenib sensitizes hepatocellular carcinoma cells to taxol by suppressing the HURP protein by Tzu-Ching Kuo; Hsing-Pang Lu; Chuck C.-K. Chao (pp. 184-194).

Sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, could inhibit HURP expression and sensitized HCC cells to sub-lethal doses of taxol through reduced translation and inhibition of the nuclear translocation of c-Rel.The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; DAPI; 4′,6-diamidino-2-phenylindole; DFF/ICAD; DNA fragmentation factor/inhibitor of caspase activated DFF; FBS; fetal bovine serum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GFP; green fluorescent protein; HCC; hepatocellular carcinoma; HURP; hepatoma upregulated protein; eIF4E; eukaryotic translation initiation factor 4E; Luc; luciferase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS; phosphate-buffered saline; PCR; polymerase chain reaction; PVDF; polyvinylidene fluoride; qRT-PCR; quantitative real-time reverse transcription-PCR; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; shRNA; short-hairpin RNAHepatocellular carcinoma; HURP; Sorafenib; Taxol resistance

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Biochemical Pharmacology (v.82, #2)

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Biochemical Pharmacology (v.82, #2)