RESUMEN
The p53 tumor suppressor plays a critical role in cancer, and more than 50% of human tumors contain mutations or deletions of the TP53 gene. p53 can transactivate or repress target genes in response to diverse stress signals, such as transient growth arrest, DNA repair, cellular differentiation, senescence and apoptosis. Through an unbiased genome-wide ChIP-seq analysis, we have found that 5-lipoxygenase (ALOX5, 5-LO) which is a key enzyme of leukotriene (LT) biosynthesis, is a direct target gene of p53 and its expression is induced by genotoxic stress via actinomycin D (Act.D) or etoposide (Eto) treatment. 5-LO and LTs play a role in immunological diseases as well as in tumorigenesis and tumor growth. p53 binds to a specific binding site consisting of a complete p53 consensus-binding motif in ALOX5 intron G which is located about 64kbp downstream of the transcriptional start site. We confirmed the strong binding of p53 to the 5-LO target site in ChIP-qPCR experiments. Expression analyses by qRT-PCR and immunoblot further revealed that genotoxic stress induces the ALOX5 mRNA and protein expression in a p53-dependent manner. Knockdown of p53 in U2OS cells leads to a downregulation of 5-LO mRNA and protein expression. In addition, immunofluorescence and immunoprecipitation assays indicate the direct binding of 5-LO to p53 protein. Furthermore, we found that 5-LO can inhibit the transcriptional activity of p53 suggesting that 5-LO acts in a negative feedback loop to limit induction of p53 target genes.
Asunto(s)
Araquidonato 5-Lipooxigenasa/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteína p53 Supresora de Tumor/fisiología , Sitios de Unión , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Dactinomicina/farmacología , Etopósido/farmacología , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
(Z)-3,5,4'-Trimethoxystilbene (Z-TMS) is a resveratrol analog with increased antiproliferative activity towards a number of cancer cell lines compared to resveratrol, which has been shown to inhibit tubulin polymerization in vitro. The purpose of this study was to investigate if Z-TMS still shows potential for the prevention of metabolic diseases as known for resveratrol. Cell growth inhibition was determined with IC50 values for Z-TMS between 0.115µM and 0.473µM (resveratrol: 110.7µM to 190.2µM). Flow cytometric analysis revealed a G2/M arrest after Z-TMS treatment, whereas resveratrol caused S phase arrest. Furthermore, Z-TMS was shown to impair microtubule polymerization. Beneficial effects on lipid accumulation were observed for resveratrol, but not for Z-TMS in an in vitro steatosis model. (E)-Resveratrol was confirmed to elevate cAMP levels, and knockdown of AMPK attenuated the antiproliferative activity, while Z-TMS did not show significant effects in these experiments. SIRT1 and AMPK activities were further measured indirectly via induction of the target gene small heterodimer partner (SHP). Thereby, (E)-resveratrol, but not Z-TMS, showed potent induction of SHP mRNA levels in an AMPK- and SIRT1-dependent manner, as confirmed by knockdown experiments. We provide evidence that Z-TMS does not show beneficial metabolic effects, probably due to loss of activity towards resveratrol target genes. Moreover, our data support previous findings that Z-TMS acts as an inhibitor of tubulin polymerization. These findings confirm that the methylation of resveratrol leads to profound changes in the mode of action, which should be taken into consideration when conducting lead structure optimization approaches.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Neoplasias Hepáticas/patología , Estilbenos/farmacología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Antineoplásicos/química , Células CACO-2 , Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica , Células HT29 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Estructura Molecular , Interferencia de ARN , Resveratrol , Sirtuina 1/genética , Sirtuina 1/metabolismo , Estilbenos/química , Relación Estructura-Actividad , Transfección , Moduladores de Tubulina/farmacologíaRESUMEN
The objective of this study was to investigate, whether the naturally occurring polyphenol resveratrol (Res) enhances the anti-tumor activities of the chemotherapeutic agent oxaliplatin (Ox) in a cell culture model of colorectal cancer, also with regard to a possible inflammatory response and cytotoxic side-effects. Res and Ox in combination synergistically inhibit cell growth of Caco-2 cells, which seems to be due to the induction of different modes of cell death and further leads to an altered cytokine profile of cocultured macrophages. Moreover, combinatorial treatment does not affect non-transformed cells as severe cytotoxicity is not detected in human foreskin fibroblasts and platelets.