RESUMEN
BACKGROUND/AIM: An in vitro cell model of long-term estrogen-deprived MCF-7 (LTED) cells has been utilized to analyze the re-growth mechanisms of breast cancers treated with blockers for estrogen receptor α (ERα) signaling. Bongkrekic acid (BKA) is a natural toxin isolated from coconut tempeh contaminated with the bacterium Burkholderia cocovenans. MATERIALS AND METHODS: LTED cells, MCF-7 cells and MDA-MB-231 cells were employed in the study. After treatment with BKA (chemically synthesized; purity: >98%), several biochemical analyses were carried out. RESULTS: LTED cells were categorized into an oxidative phenotype. When LTED cells were treated with BKA, lactate dehydrogenase A (LDH-A)/pyruvate dehydrogenase kinase 4 (PDK4) were down-regulated, thereby prompting the aggressive use of glucose via mitochondrial oxidative phosphorylation and induction of cell death responses. These effects of BKA were not observed in the other breast cancer cells analyzed. CONCLUSION: We suggest the potential of BKA as an experimental tool for the analysis of cancer biology in LTED cells.
Asunto(s)
Ácido Bongcréquico/farmacología , Neoplasias de la Mama/metabolismo , Antígenos de Neoplasias/genética , Carnitina O-Palmitoiltransferasa/genética , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Estradiol , Femenino , Glucosa/metabolismo , Humanos , Isoenzimas/genética , Antígeno Ki-67/genética , L-Lactato Deshidrogenasa/genética , Lactato Deshidrogenasa 5 , Mitocondrias/metabolismo , PPAR gamma/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
Previously, we reported that (-)-xanthatin, a naturally occurring xanthanolide present in the Cocklebur plant, exhibits potent anti-proliferative effects on human breast cancer cells, accompanied by an induction of the growth arrest and DNA damage-inducible gene 45γ (GADD45γ), recognized recently as a novel tumor suppressor gene. However, the mechanisms mediating this activation were unknown. Topoisomerase IIα (Topo IIα) inhibition has been reported to produce a cell death response accompanied by an atypical DNA laddering fragmentation profile, similar to that noted previously for (-)-xanthatin. Therefore we hypothesized that (-)-xanthatin's GADD45γ activation was mediated through the Topo IIα pathway. Here, we identify that (-)-xanthatin does function as a catalytic inhibitor of Topo IIα, promoting DNA damage. In addition, reactive oxygen species (ROS) were elevated in cells treated with this agent. Mechanistically, it was determined that the induced levels of GADD45γ mRNA resulting from (-)-xanthatin exposures were stabilized by coordinately produced ROS, and that the consequent induction of GADD45γ mRNA, GADD45γ protein and ROS generation were abrogated by co-treatment with N-acetyl-l-cysteine. Taken together, the data support the concept that Topo IIα inhibition by (-)-xanthatin is a trigger that stimulates expression of DNA damage-inducible GADD45γ mRNA and that concomitantly produced ROS act downstream to further enhance the GADD45γ mRNA/GADD45γ protein induction process, resulting in breast cancer cell death.
Asunto(s)
Antígenos de Neoplasias/fisiología , ADN-Topoisomerasas de Tipo II/fisiología , Proteínas de Unión al ADN/fisiología , Furanos/farmacología , Insecticidas/farmacología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Inhibidores de Topoisomerasa II , Acetilcisteína/farmacología , Antígenos de Neoplasias/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Daño del ADN , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Femenino , Depuradores de Radicales Libres/farmacología , Glutatión/metabolismo , Semivida , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Regulación hacia Arriba/efectos de los fármacos , Proteinas GADD45RESUMEN
15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein (ox-LDL), a major causal factor for atherosclerosis. Both enzymatic (15-LOX) and non-enzymatic (Cu(2+)) mechanisms have been proposed for the production of ox-LDL. We have recently reported that cannabidiol-2',6'-dimethyl ether (CBDD) is a selective and potent inhibitor of 15-LOX-catalyzed linoleic acid oxygenation (Takeda et al., Drug Metab. Dispos., 37, 1733-1737 (2009)). In the LDL, linoleic acid is present as cholesteryl linoleate, the major fatty acid esterified to cholesterol, and is susceptible to oxidative modification by 15-LOX or Cu(2+). In this investigation, we examined the efficacy of CBDD on i) 15-LOX-catalyzed oxygenation of cholesteryl linoleate, and ii) ox-LDL formation catalyzed by 15-LOX versus Cu(2+)-mediated non-enzymatic generation of this important mediator. The results obtained demonstrate that CBDD is a potent and selective inhibitor of ox-LDL formation generated by the 15-LOX pathway. These studies establish CBDD as both an important experimental tool for characterizing 15-LOX-mediated ox-LDL formation, and as a potentially useful therapeutic agent for treatment of atherosclerosis.
