The Combination of Interferon-Beta and HMG-CoA Reductase Inhibition in Multiple Sclerosis: Enthusiasm Lost too Soon?

Recent studies support the notion that statins, widely prescribed cholesterol-lowering agents, may target key elements in the immunological cascade leading to inflammation and tissue damage in the pathogenesis of multiple sclerosis (MS). Compelling experimental and observational clinical studies highlighted the possibility that statins may also exert immunomodulatory synergy with approved MS drugs, resulting in several randomized clinical trials testing statins in combination with interferon-beta (IFN-?). Some data, however, suggest that this particular combination may not be clinically beneficial, and might actually have a negative effect on the disease course in some patients with MS. In this regard, a small North American trial indicated that atorvastatin administered in combination with IFN-? may increase disease activity in relapsing-remitting MS. Although other trials did not confirm this finding, the enthusiasm for studies with statins dwindled. This review aims to provide a comprehensive overview of the completed clinical trials and reports of the interim analyses evaluating the combination of IFN-? and statins in MS. Moreover, we try to address the evident question whether usage of this combination routinely requires caution, since the number of IFN-?-treated MS patients receiving statins for lowering of cholesterol is expected to grow.

High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer

Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer.

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

High expression of octamer-binding transcription factor 4A, prominin-1 and aldehyde dehydrogenase strongly indicates involvement in the initiation of lung adenocarcinoma resulting in shorter disease-free intervals

The increasing relevance of the cancer stem cell (CSC) hypothesis and the impact of CSC-associated markers in the carcinogenesis of solid tumours may provide potential prognostic implications in lung cancer. We propose that a collective genetic analysis of established CSC-related markers will generate data to better define the role of putative CSCs in lung adenocarcinoma (LAC).

Quantification of oxidized levels of specific RNA species using an aldehyde reactive probe

Emerging evidence has shown that oxidation of RNA, including messenger RNA (mRNA), is elevated in several age-related diseases, although investigation of oxidized levels of individual RNA species has been limited. Recently we reported that an aldehyde reactive probe (ARP) quantitatively reacts with oxidatively modified depurinated/depyrimidinated (abasic) RNA. Here we report a novel method to isolate oxidized RNA using ARP and streptavidin beads. An oligo RNA containing abasic sites that were derivatized with ARP was pulled down by streptavidin beads, whereas a control oligo RNA was not. In vitro oxidized RNA, as well as total cellular RNA, isolated from oxidatively stressed cells was also pulled down, dependent on oxidation level, and concentrated in the pull-down fraction. Quantitative reverse transcription polymerase chain reaction (RT-PCR) using RNA in the pull-down fraction demonstrated that several gene transcripts were uniquely increased in the fraction by oxidative stress. Thus, our method selectively concentrates oxidized RNA by pull-down and enables the assessment of oxidation levels of individual RNA species. (C) 2011 Elsevier Inc. All rights reserved.

Reductase inhibitory activity of some flavones: Topological descriptors in modeling the activity

In healthy people, glucose is metabolized through Embden-Meyerhoff pathway. In cases of diabetes mellitus, with the increased levels of glucose in insulin-insensitive tissues the Aldose Reductase (AR) in polyol pathway facilitates the conversion of glucose to sorbitol. In this cascade of events the accumulated sorbitol is attributed to be responsible for cataract, neuropathy and retinopathy in diabetic cases.1,2 Thus, the inhibition of AR in polyol pathway may prevent and lead to the cure of the complications arising out of the diabetes mellitus. In this background, Matsuda and coworkers3 studied the AR inhibitory activity of large number of flavones and related compounds from traditional antidiabetic remedies. Here, many of these compounds shared 2-Aryl-benzpyran-4-one as scaffold for different chemical groups surrounding this moiety. This offers scope to investigate the AR inhibitory activity of these compounds in relation to the functional group environment surrounding this core

In vitro incorporation of molybdate into demolybdoproteins in Escherichia coli.

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.

Effects of gossypol on DNA replication in mammalian cells

Gossypol, a binaphthalene compound, possesses male infertility effects. However, its mechanism of action and effects on somatic cells are not yet understood. The purpose of this study was to examine the effects of gossypol on mammalian cell growth and DNA replication, using tissue culture cells (HeLa) as an in vivo model.^ Gossypol inhibited DNA synthesis in HeLa cells at low doses, without affecting RNA or protein synthesis. This caused cells to accumulate in S phase without affecting cells in other phases of the cell cycle. The inhibition of DNA synthesis was both dose- and time-dependent. This irreversible block was associated with a decrease in HeLa plating efficiency. Gossypol did bind to DNA but did not measurably affect its ability to serve as a template for DNA polymerase $\alpha$, the major replicative enzyme. Only in the absence of serum could gossypol induce single-strand DNA breaks in HeLa cells; no DNA-DNA or DNA-protein crosslinks were formed.^ Gossypol exhibited dose-dependent inhibition of a number of eukaryotic and prokaryotic replicative DNA polymerases both in vitro and in vivo. This inhibition was kinetically non-competitive with respect to the DNA template and dNTP substrates. Both a filter binding assay and polyacrylamide gel electrophoresis were used to study gossypol binding to DNA polymerase. Inhibition resulted from drug binding to two adjacent amino acid residues on the enzyme. Binding was found to be irreversible and mediated through either non-covalent interactions or by Schiff's base formation between the aldehyde groups of gossypol and the $\varepsilon$-NH$\sb2$ groups of amino acid residues on the polymerase. Structure-function studies using eleven gossypol derivatives revealed that both aldehyde and hydroxyl groups function independently to effect inhibition of DNA polymerase and DNA replication. The activities of DNA polymerase $\beta$ and ribonucleotide reductase were also inhibited by increasing gossypol concentrations.^ These studies demonstrate that the gossypol-mediated inhibition of DNA replication is due in part to inhibition of key replicative enzymes, such as DNA polymerase $\alpha$. The study of DNA polymerase may serve as a model for the interaction of enzymes with gossypol, a drug which may prove useful as a chemotherapeutic agent. ^

Studies on the interaction of NADPH cytochrome P-450 reductase and cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the K$\sb{\rm m}$ of the reductase for cytochrome P-450b or cytochrome P-450c. Modification of carboxyl groups on the reductase with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, and taurine, respectively. The apparent K$\sb{\rm m}$ for cytochrome P-450c or cytochrome P-450b was increased 1.3 to 5.2 fold. There were varied effects on the V$\sb{\rm max}$. There was no significant change in the conformation of the reductase upon chemical modification. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450. Cytochrome P-450 protected 0.8 moles of carboxyl residues on the reductase from being modified with EDC. These protected amino acids on the reductase are presumably involved in binding to cytochrome P-450. The specific peptide containing these amino acids has been identified. ^

The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

Cytochrome P-450 reductase FAD binding domain: Cloning, enzymatic activity and flavin binding characterization

The Mixed Function Oxidase System metabolizes a wide range of biochemicals including drugs, pesticides and steroids. Cytochrome P450 reductase is a key enzymatic component of this system, supplying reducing equivalents from NADPH to cytochrome P450. The electrons are shuttled through reductase via two flavin moieties: FAD and FMN. Although the exact mechanism of flavins action is not known, the enzymatic features of reductase greatly depleted of either FMN of FAD have been characterized. Additionally, flavin location within reductase has been proposed by homology and chemical modification studies. This study seeks to extend the flavin depletion analysis in a more controlled system by eliminating the proposed FMN binding domain with recombinant DNA techniques and biochemical analysis. Two P450 reductase cDNA clones containing only the FMN and NADPH binding domain were isolated, expressed and the protein products purified and analysed. This study confirms the proposed FAD binding site, role of FAD in electron shuttling pathway and provides new methods to study the FAD binding domain. ^