RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Cisplatino/farmacología , Neoplasias del Colon/tratamiento farmacológico , Compuestos Organoplatinos/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Células HCT116 , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxaliplatino , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Lipólisis/efectos de los fármacos , Obesidad/tratamiento farmacológico , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adiponectina/genética , Animales , Citoesqueleto/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Ingestión de Energía , Perfilación de la Expresión Génica , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Fenotipo , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Rimonabant , Termogénesis/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Ciclohexanos/farmacología , Neoplasias de la Próstata/patología , Transcripción Genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Humanos , Ligandos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores sigma , Timidina Quinasa/antagonistas & inhibidores , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Diferenciación Celular , Receptores de GABA-A/metabolismo , Linfocitos T/metabolismo , Timo/citología , Apoptosis , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células Jurkat , Activación de Linfocitos , Microscopía Confocal , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Linfocitos T/citologíaRESUMEN
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.
Asunto(s)
Ciclohexanos/farmacología , Regulación Fúngica de la Expresión Génica , Inmunosupresores/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Saccharomyces cerevisiae/efectos de los fármacos , Northern Blotting , División Celular , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Canal de Potasio ERG1 , Ergosterol/metabolismo , Canales de Potasio Éter-A-Go-Go , Cromatografía de Gases y Espectrometría de Masas , Modelos Biológicos , Hibridación de Ácido Nucleico , ARN/metabolismo , ARN Mensajero , Transactivadores/metabolismo , Regulador Transcripcional ERG , Regulación hacia ArribaRESUMEN
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.