Repression of cell cycle-related proteins by oxaliplatin but not cisplatin in human colon cancer cells.

Oxaliplatin (Eloxatin) is a third-generation platinum derivative with an in vitro and in vivo spectrum of activity distinct from that of cisplatin, especially in colon cancer cells. Here, we studied the molecular basis of this difference on the HCT-116 human colon carcinoma cell line (mismatch repair-deficient, wild-type functional p53). Oxaliplatin inhibited HCT-116 cell proliferation with greater efficacy than cisplatin. At comparable concentrations, cisplatin slowed down the replication phase and activated the G2-M checkpoint, whereas oxaliplatin activated the G1-S checkpoint and completely blocked the G2-M transition. With the aim of finding oxaliplatin-specific target genes and mechanisms differing from those of cisplatin, we established the transcriptional signatures of both products on HCT-116 cells using microarray technology. Based on hierarchical clustering, we found that (a) many more genes were modulated by oxaliplatin compared with cisplatin and (b) among the 117 modulated genes, 79 were regulated similarly by both drugs and, in sharp contrast, 38 genes were dose dependently down-regulated by oxaliplatin and, conversely, up-regulated or unaffected by cisplatin. Interestingly, several cell cycle-related genes encoding proteins involved in DNA replication and G2-M progression belong to this latter group. RNA modulations, confirmed at the protein level, were in accordance with oxaliplatin- and cisplatin-induced cell cycle variations. Beyond the identification of genes affected by both drugs, the identified oxaliplatin-specific target genes could be useful as predictive markers for evaluating and comparing the efficacy and molecular pharmacology of platinum drugs.

The CB1 receptor antagonist rimonabant reverses the diet-induced obesity phenotype through the regulation of lipolysis and energy balance.

We investigated the molecular events involved in the long-lasting reduction of adipose mass by the selective CB1 antagonist, SR141716. Its effects were assessed at the transcriptional level both in white (WAT) and brown (BAT) adipose tissues in a diet-induced obesity model in mice. Our data clearly indicated that SR141716 reversed the phenotype of obese adipocytes at both macroscopic and genomic levels. First, oral treatment with SR141716 at 10 mg/kg/d for 40 days induced a robust reduction of obesity, as shown by the 50% decrease in adipose mass together with a major restoration of white adipocyte morphology similar to lean animals. Second, we found that the major alterations in gene expression levels induced by obesity in WAT and BAT were mostly reversed in SR141716-treated obese mice. Importantly, the transcriptional patterns of treated obese mice were similar to those obtained in the CB1 receptor knockout mice fed a high-fat regimen and which are resistant to obesity, supporting a CB1 receptor-mediated process. Functional analysis of these modulations indicated that the reduction of adipose mass by the molecule resulted from an enhanced lipolysis through the induction of enzymes of the beta-oxidation and TCA cycle, increased energy expenditure, mainly through futile cycling (calcium and substrate), and a tight regulation of glucose homeostasis. These changes accompanied a significant cellular remodeling and contributed to a reduction of the obesity-related inflammatory status. In addition to a transient reduction of food consumption, increases of both fatty acid oxidation and energy expenditure induced by the molecule summate leading to a sustained weight loss. Altogether, these data strongly indicate that the endocannabinoid system has a major role in the regulation of energy metabolism.

Expression of the peripheral benzodiazepine receptor triggers thymocyte differentiation.

In the thymus, during T-cell differentiation, the expression of the peripheral benzodiazepine receptor (PBR) modulates. The protein level decreases between the double negative and double positive stages, and then increases when thymocytes become single positive. We addressed the role played by PBR in T-cell maturation. To this aim, we used Jurkat cells, which are immature T lymphocytes derived from an acute lymphoblastic leukemia. These cells are PBR negative and were stably transfected to achieve PBR levels similar to that in mature T cells. Using the DNA chip technology, we analyzed the PBR expression-dependent gene changes and evidenced that PBR-expressing cells exhibited more mature features than mock-transfected ones. A majority of the modulated genes encode proteins playing direct or indirect roles during the lymphocyte maturation process. In particular, PBR expression induced several differentiation markers (such as CD1, CD6), or key regulating elements (e.g., RAG1, RAG2, CD99, TCR). By contrast, some regulators of TCR signaling were reduced. PBR expression also affected the expression of critical apoptosis regulators: the proapoptotic lipocortin I, galectin-1, and galectin-9 were reduced while the antiapoptotic Bcl-2 was induced. Altogether our results supported the hypothesis that PBR controls T-cell maturation and suggested mechanisms through which PBR may regulate thymocyte-positive selection.

SR31747A: a peripheral sigma ligand with potent antitumor activities.

SR31747A is currently being evaluated in phase IIb clinical trials for prostate cancer treatment. The molecule is a peripheral sigma ligand that binds four proteins in human cells, i.e. SRBP-1, sigma-2, HSI and its relative SRBP-2. SR31747A is a dual agent with both immunomodulatory and antiproliferative activities. The molecule blocks proliferation of human and mouse lymphocytes, modulates the expression of pro- and anti-inflammatory cytokines, and was shown to protect animals in vivo against acute and chronic inflammatory conditions such as acute graft-versus-host reaction, lethality induced by staphylococcal enterotoxin B and lipopolysaccharide or rheumatoid arthritis. Besides these immunomodulatory activities, the molecule also inhibits the proliferation of various tumor cell lines in vitro in a time- and concentration-dependent manner. In vivo, SR31747A has potent antitumoral activity as demonstrated against mammary and prostatic tumoral cell lines injected into nude mice, where both tumor incidence and growth were decreased by more than 40% following daily SR31747A treatment at 25 mg/kg i.p. The recent literature on SR31747A in cancer is reviewed here. We focus specifically on preclinical data obtained in vivo and on studies aimed at deciphering the mode of action of the molecule.

Immunohistochemical assessment of the peripheral benzodiazepine receptor in breast cancer and its relationship with survival.

PURPOSE: The peripheral benzodiazepine receptor (PBR) expression has been shown dramatically increased in neoplastic tissues and tumor cell lines originated from ovary, liver, colon, breast, or brain relative to untransformed tissues. Its expression has been also associated with tumor progression and aggressiveness. To explore whether PBR expression level could be of prognostic value in invasive breast cancer, we studied a series of 117 patients who underwent surgery for primary breast carcinomas and were followed-up for 8 years. EXPERIMENTAL DESIGN: Using an immunohistochemical approach, we first compared PBR expression in normal and tumoral tissues, then we studied PBR expression together with clinicopathological variables (histological type, histological grade, lymph node, estrogen and progesterone receptor status), and biological markers such as BclII, Ki-67, and HER2/Neu. RESULTS: Our results revealed a significant increase of PBR expression in tumoral versus normal breast cells. We found a negative correlation between PBR expression and estrogen receptor status (P = 0.03) as well as a positive correlation between PBR and Ki-67 (P = 0.044). Although the disease-free survival was not affected by PBR in the whole population, high PBR expression level was significantly correlated with a shorter disease-free survival in the lymph node-negative patients, P = 0.038. CONCLUSIONS: As the axillary lymph node-negative status is generally considered as a good prognosis factor, the high expression of PBR in this patient subgroup may be used to identify a new high risk population, for which a more specific therapy would be beneficial.

Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues.

Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.

Transcriptomic classification of antitumor agents: application to the analysis of the antitumoral effect of SR31747A.

SR31747A is a sigma ligand that exhibits a potent antitumoral activity on various human tumor cell lines both in vitro and in vivo. To understand its mode of action, we used DNA microarray technology combined with a new bioinformatic approach to identify genes that are modulated by SR31747A in different human breast or prostate cancer cell lines. The SR31747A transcriptional signature was also compared with that of seven different representative anticancer drugs commonly used in the clinic. To this aim, we performed a two-dimensional hierarchical clustering analysis of drugs and genes which showed that 1) standard molecules with similar mechanism of action clustered together and 2) SR31747A does not belong to any previously characterized class of standard anticancer drugs. Moreover, we showed that 3) SR31747A mainly exerted its antiproliferative effect by inhibiting the expression of genes playing a key role in DNA replication and cell cycle progression. Finally, contrasting with other drugs, we obtained evidence that 4) SR31747A strongly inhibited the expression of three key enzymes of the nucleotide synthesis pathway (i.e., dihydrofolate reductase, thymidylate synthase, and thymidine kinase) with the latter shown both at the mRNA and protein levels. These results, obtained through a novel molecular approach to characterize and compare anticancer agents, showed that SR31747A exhibits an original mechanism of action, very likely through unexpected targets whose modulations may account for its antitumoral effect.

Identification and pharmacological characterization of SRBP-2: a novel SR31747A-binding protein.

SR31747A is a sigma ligand with potent antiproliferative activity against tumor cells and for which three binding proteins have been identified to date: (a) SRBP-1 (also called sigma 1); (b) HIS; and (c) sigma 2. In this study, we characterized an additional SR31747A binding site, i.e., SRBP-2 (SR31747A-binding protein 2). Using an in silico screening approach, we identified this novel sequence, which exhibits 41% homology with HSI. The 1142-bp cDNA was found to encode a 206 amino acid protein not related to SRBP-1. Northern blot analysis of SRBP-2 mRNA expression revealed a single 1.1-kb transcript that was widely expressed in organs; the liver was particularly enriched, and the brain showed the lowest abundance. A murine homologue that exhibited a similar expression pattern was also characterized. Subcellular localization analysis using specific polyclonal antibodies revealed that SRBP-2 had the same nuclear membrane and endoplasmic reticulum localization as other members of the SR31747A-binding protein family. Considering SRBP-2-binding properties, pharmacological analysis clearly highlighted that SRBP-2 was distinct from sigma 2. Scatchard plot analysis revealed K(d) values of 10 and 3 nM for SR31747A and Tamoxifen, respectively. In contrast with HSI, the protein also did not exhibit detectable isomerase activity. When analyzing SRBP-2 expression in human breast cancer biopsies, we obtained evidence that SRBP-2 expression, together with SRBP-1 and HSI, may be of interest as a prognostic marker. These findings demonstrated that SRBP-2 represents an additional molecular target for SR31747A, which could help to understand the immunosuppressive and antiproliferative effects of the molecule.

The peripheral benzodiazepine receptor: a promising therapeutic drug target.

The peripheral benzodiazepine receptor (PBR) is a critical component of the mitochondrial permeability transition pore (MPTP), a multiprotein complex located at the contact site between inner and outer mitochondrial membranes, which is intimately involved in the initiation and regulation of apoptosis. PBR is a small evolutionary conserved protein, located at the surface of the mitochondria where it is physically associated with the voltage-dependent anion channel (VDAC) and adenosine nucleotide translocase (ANT) that form the backbone of MPTP. PBR is widely distributed throughout the body and has been associated with numerous biological functions. Consistent with its localization in the MPTP, PBR is involved in the regulation of apoptosis, but also in the regulation of cell proliferation, stimulation of steroidogenesis, immunomodulation, porphyrin transport, heme biosynthesis, anion transport and regulation of mitochondrial functions. The recent literature on PBR is reviewed here. Specifically, we highlight numerous results suggesting that the use of specific PBR ligands to modulate PBR activity may have potential therapeutic applications and might be of significant clinical benefit in the management of a large spectrum of different indications including cancer, auto-immune, infectious and neurodegenerative diseases. In addition, we present the proposed mechanisms by which the molecules exerted these effects, particularly oriented on the modulation of the MPTP activities.

Involvement of the peripheral benzodiazepine receptor in the development of cutaneous pathology in Mrl/Lpr mice.

Mrl/Lpr mice develop inflammatory pathologies similar to human lupus erythematosus (LE). In that model, we showed a protective effect of different peripheral benzodiazepine receptor (PBR) ligands: PK 11195, Ro5-4864 and the newly described SSR180575 on the development of the cutaneous lesions. Specifically, we evidenced that a chronic treatment at 3 mg/kg per i.p. for 30 days prevented acanthosis, hyperkeratosis and generation of dermal infiltrates as compared with control untreated mice. In addition, using a specific polyclonal anti mouse PBR antibody, we characterized PBR expression in the skin lesions, and we observed that PBR expression in the epidermal component was increased when Mrl/Lpr mice developed the pathology and diminished upon PBR ligand treatment. PBR expression modulation together with the protective effects of its ligands further reinforce the role that PBR may play in the regulation of inflammation processes. Provided the exact mechanism of action that accounts for PBR action in that process is elucidated, these data support new therapeutic applications for specific potent PBR ligands.

Expression profile and up-regulation of PRAX-1 mRNA by antidepressant treatment in the rat brain.

A protein associated with the peripheral-type benzodiazepine receptor (PRAX-1) has recently been cloned, but its regional distribution in the central nervous system and its function remain to be clarified. In situ hybridization was carried out to localize PRAX-1 mRNA in the rat brain and revealed a high expression of the transcript in limbic structures such as the CA1 region of the hippocampus, as well as the dentate gyrus, septum, amygdala, and the islands of Calleja. A dense hybridization signal was also observed in the nucleus accumbens, caudate nucleus, olfactory tubercle, pineal gland, and cerebellar cortex. PRAX-1 mRNA expression was largely neuronal; it colocalized with neuron-specific enolase but not glial fibrillary acidic protein. Long-term treatments (21 days) with the neuroleptic haloperidol increased PRAX-1 mRNA expression only in the dentate gyrus, whereas anxiolytic/anticonvulsant diazepam had no effect in any of the hippocampal region studied. Repeated electroconvulsive shock administration significantly enhanced PRAX1 expression in the CA1 subfield and dentate gyrus. Several classes of antidepressant treatment, including serotonin selective reuptake inhibitor (fluoxetine), mixed serotonin- and norepinephrine-uptake inhibitor (imipramine), and monoamine oxidase inhibitors (iproniazid and tranylcypromine), shared this effect. Furthermore, the selective nonpeptide NK2 receptor antagonist (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR48968), which shows an antidepressant profile in animal studies, also enhanced PRAX-1 mRNA expression. These results point to a potential role of PRAX-1 function in the central nervous system and suggest that the up-regulation of PRAX-1 mRNA represents a common action of chronic antidepressant treatment.

A DNA microarray-based approach to elucidate the effects of the immunosuppressant SR31747A on gene expression in Saccharomyces cerevisiae.

SR31747A is an immunosuppressive agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae. In this microorganism, SR31747A was shown to inhibit the ERG2 gene product, namely the delta8-delta7 sterol isomerase, involved in the ergosterol biosynthesis pathway. Although previous genetic experiments pointed to this enzyme as the target for SR31747A in yeast, the existence of other potential targets could not be ruled out. To enlighten this issue, we undertook a DNA microarray-based approach in which the expression profile of SR31747A-treated wild-type cells defining the "drug signature" was compared with the "mutant signature," the expression profile of the corresponding ERG2-deleted strain. We observed that treatment of ERG2-positive cells with SR31747A resulted in the modulation of mRNA levels of numerous genes. Among them, 121 werealso affected in untreated ERG2-disrupted cells compared with wild-type cells. By contrast, drug exposure did not induce any significant transcriptional change in the ERG2 null mutant. These results were consistent with SR31747A being an inhibitor of the sterol isomerase and demonstrated the absence of any additional SR31747A target. The detailed analysis of the observed 121 modulated genes provides new insights into the cellular response to ergosterol deprivation induced by SR31747A through inhibition of the ERG2 gene product.

SSR125329A, a high affinity sigma receptor ligand with potent anti-inflammatory properties.

SSR125329A ([(Z)-3-(4-Adamantan-2-yl-3,5-dichloro-phenyl)-allyl]-cyclohexyl-ethyl-amine) is a new ligand exhibiting high affinity for sigma(1) and sigma(2) receptors and for the human Delta8-Delta7-sterol isomerase. Here we show that this molecule has potent immunoregulatory properties both in vitro and in vivo. SSR125329A inhibited staphylococcal enterotoxin B-induced mouse splenocyte proliferation in vitro, whereas in vivo it enhanced lipopolysaccharide-induced systemic release of interleukin-10 while simultaneously inhibiting tumor necrosis factor-alpha (TNF-alpha) synthesis. It also prevented graft-versus-host disease in B6D2F1 mice and protected Mrl/lpr mice against the development of its spontaneous rheumatoid-like syndrome. There is high interplay of pro- and anti-inflammatory cytokines in inflammatory processes, particularly in human rheumatoid arthritis. The results of this study provide substantial evidence that sigma receptor ligands may represent a new effective approach for rheumatoid arthritis treatment.

Involvement of the peripheral benzodiazepine receptor in the development of rheumatoid arthritis in Mrl/lpr mice.

In this study, the effects of different peripheral benzodiazapine receptor ligands: PK 11195 [1-(2-chloro-phenyl)-N-methyl-N-(1-methylpropyl)-1-isoquinoline carboxamide], Ro5-4864 [7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one] and the newly described SSR 180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridozine[4,5-b] indole-1-acetamide) were analysed on the progression and severity of rheumatoid arthritis in vivo in the Mrl/lpr mice model, following chronic treatment (at 3 mg/kg, i.p. for 30 days). We found that peripheral benzodiazepine receptor ligands have significant beneficial therapeutic action on the development of spontaneous rheumatoid arthritis-like signs. Concomitantly, we mapped immunoreactive peripheral benzodiazepine receptor in inflamed tissues, and we observed that in addition to the infiltrated leukocytes, peripheral benzodiazepine receptor was expressed in synovial membranes, at the cartilage pannus junction and in chondrocytes. Interestingly, we observed that peripheral benzodiazepine receptor expression in chondrocytes was reduced when Mrl/lpr mice developed the pathology and restored upon peripheral benzodiazepine receptor ligand treatment. Altogether, our data provide further evidence of a role played by peripheral benzodiazepine receptor in the regulation of inflammation processes and support new therapeutic applications for specific potent peripheral benzodiazepine receptor ligands.

Peripheral benzodiazepine receptors and mitochondrial function.

For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.