RESUMEN
4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A ß-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.
Asunto(s)
Aldehídos/química , Aldehídos/farmacología , Portadores de Fármacos/química , Lípidos/química , Melanoma/patología , Nanocápsulas/química , Acrilamidas/química , Transporte Biológico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclodextrinas/química , Estabilidad de Medicamentos , Humanos , Morfolinas/químicaRESUMEN
4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, ß-cyclodextrin (ßCD) polymers are playing a very important role in drug formulation by virtue of the ability of ßCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several ßCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of ßCD, the ßCD-poly(4-acryloylmorpholine) conjugate (PACM-ßCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-ßCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.
Asunto(s)
Aldehídos/farmacología , Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , beta-Ciclodextrinas/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Humanos , Concentración 50 InhibidoraRESUMEN
PPARs are nuclear receptors activated by ligands. Activation of PPARγ leads to a reduction of adhesion and motility in some cancer models. PPARγ transcriptional activity can be negatively regulated by JNK-mediated phosphorylation. We postulated that the use of agents able to inhibit JNK activity could increase the effectiveness of PPARγ ligands. We analysed the effects of rosiglitazone (PPARγ ligand) and AS601245 (a selective JNK inhibitor) alone or in association on adhesion and migration of CaCo-2, HT29, and SW480 human colon cancer cells and investigated, through microarray analysis, the genes involved in these processes. Cell adhesion and migration was strongly inhibited by rosiglitazone and AS601245. Combined treatment with the two compounds resulted in a greater reduction of the adhesion and migration capacity. Affymetrix analysis in CaCo-2 cells revealed that some genes which were highly modulated by the combined treatment could be involved in these biological responses. Rosiglitazone, AS601245 and combined treatment down-regulated the expression of fibrinogen chains in all three cell lines. Moreover, rosiglitazone, alone or in association with AS601245, caused a decrease in the fibrinogen release. ARHGEF7/ß-PIX gene was highly down-regulated by combined treatment, and western blot analysis revealed that ß-PIX protein is down-modulated in CaCo-2, HT29 and SW480 cells, also. Transfection of cells with ß-PIX gene completely abrogated the inhibitory effect on cell migration, determined by rosiglitazone, AS601245 and combined treatment. Results demonstrated that ß-PIX protein is involved in the inhibition of cell migration and sustaining the positive interaction between PPARγ ligands and anti-inflammatory agents in humans.
Asunto(s)
Acetonitrilos/farmacología , Benzotiazoles/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/patología , Expresión Génica/efectos de los fármacos , Tiazolidinedionas/farmacología , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/genética , Fibrinógeno/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , RosiglitazonaRESUMEN
PPARαs are nuclear receptors highly expressed in colon cells. They can be activated by the fibrates (clofibrate, ciprofibrate etc.) used to treat hyperlipidemia. Since PPARα transcriptional activity can be negatively regulated by JNK, the inhibition of JNK activity could increase the effectiveness of PPARα ligands. We analysed the effects of AS601245 (a JNK inhibitor) and clofibrate alone or in association, on proliferation, apoptosis, differentiation and the gene expression profile of CaCo-2 human colon cancer cells. Proliferation was inhibited in a dose-dependent way by clofibrate and AS601245. Combined treatment synergistically reduced cell proliferation, cyclin D1 and PCNA expression and induced apoptosis and differentiation. Reduction of cell proliferation, accompanied by the modulation of p21 expression was observed in HepG2 cells, also. Gene expression analysis revealed that some genes were highly modulated by the combined treatment and 28 genes containing PPRE were up-regulated, while clofibrate alone was ineffective. Moreover, STAT3 signalling was strongly reduced by combined treatment. After combined treatment, the binding of PPARα to PPRE increased and paralleled with the expression of the PPAR coactivator MED1. Results demonstrate that combined treatment increases the effectiveness of both compounds and suggest a positive interaction between PPARα ligands and anti-inflammatory agents in humans.
RESUMEN
4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy.
Asunto(s)
Aldehídos/metabolismo , Núcleo Celular/metabolismo , Peroxidación de Lípido , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias de la Próstata/metabolismo , Aldehídos/farmacología , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Peroxidación de Lípido/genética , Masculino , Factor 2 Relacionado con NF-E2/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Peroxisome proliferator-activated receptor-γ (PPARγ) is involved in regulation of macrophage inflammation and in atherosclerosis. Herein we investigate the influence of statin treatment on PPARγ expression in coronary artery disease. METHOD: PPARγ expression was investigated in coronary atherosclerotic atherectomies (N=48) and arteries (N=12) from patients with stable or unstable coronary syndromes or undergoing cardiac transplantation for end-stage ischemic cardiomyopathy, respectively, by immunohistochemistry. Plaque components and tissue factor immunoreactivity were also investigated. Atherectomies were obtained from de novo culprit lesions of hypercholesterolemic (16 statin-treated and 16 untreated) and normolipidemic (N=16) patients. Furthermore, PPARγ expression was evaluated in patients peripheral blood monocytes and in monocytic U937 cells after atorvastatin incubation, by Western blot analysis. RESULT: PPARγ expression was higher in coronary plaques and peripheral blood monocytes of statin-treated patients, and it significantly increased in monocytes after 24h atorvastatin incubation (p<0.05). Intra-plaque macrophage content, atheroma, neoangiogenesis and hemorrhage, and circulating CRP levels were lower in statin-treated than untreated hypercholesterolemic patients and comparable with normolipidemic subjects. PPARγ immunoreactivity was localized to neointima and media, its distribution pattern being different from that of tissue factor. CONCLUSION: PPARγ expression was enhanced in statin-treated patients with different distribution and behavior as compared to atheroma, macrophage content, tissue factor immunoreactivity and serum CRP. In vitro studies showed increased PPARγ expression in monocytes after atorvastatin incubation. These findings provide further evidence as to the protective role of statins in coronary artery disease and their influence on PPARγ expression in coronary plaques and on the inflammatory status of patients.
Asunto(s)
Enfermedad de la Arteria Coronaria/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hiperlipidemias/metabolismo , PPAR gamma/metabolismo , Adulto , Anciano , Arterias/metabolismo , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Femenino , Humanos , Hiperlipidemias/tratamiento farmacológico , Inmunohistoquímica/métodos , Inflamación , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/metabolismo , Células U937RESUMEN
Histone deacetylase inhibitors (HDACIs) are promising antineoplastic agents for the treatment of cancer. Here we report that the lipid peroxidation end product 4-hydroxynonenal (HNE) significantly potentiates the anti-tumor effects of the HDAC inhibitor panobinostat (LBH589) in the PC3 prostate cancer cell model. Panobinostat and HNE inhibited proliferation of PC3 cells and the combination of the two agents resulted in a significant combined effect. Cell cycle analysis revealed that both single agents and, to a greater extent, their combined treatment induced G2/M arrest, but cell death occurred in the combined treatment only. Furthermore, HNE and, to a greater extent, the combined treatment induced dephosphorylation of Cdc2 leading to progression into mitosis as confirmed by α-tubulin/DAPI staining and phospho-histone H3 (Ser10) analysis. To evaluate possible induction of DNA damage we utilized the marker phosphorylated histone H2A.X. Results showed that the combination of panobinostat and HNE induced significant DNA damage concomitant with the mitotic arrest. Then, by using androgen receptor (AR)-expressing PC3 cells we observed that the responsiveness to HNE and panobinostat was independent of the expression of functional AR. Taken together, our data suggest that HNE potentiates the antitumoral effect of the HDACI panobinostat in prostate cancer cells.
Asunto(s)
Aldehídos/farmacología , Ciclo Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Daño del ADN/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Acetilación , Apoptosis/efectos de los fármacos , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glutatión/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Indoles , Peroxidación de Lípido/efectos de los fármacos , Masculino , Panobinostat , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Células Tumorales CultivadasRESUMEN
In human cells the length of telomeres depends on telomerase activity. This activity and the expression of the catalytic subunit of human telomerase reverse transcriptase (hTERT) is strongly up-regulated in most human cancers. hTERT expression is regulated by different transcription factors, such as c-Myc, Mad1 and Sp1. In this study, we demonstrated that 15d-PG J2 and rosiglitazone (an endogenous and synthetic peroxisome proliferators activated receptor gamma (PPARgamma) ligand, respectively) inhibited hTERT expression and telomerase activity in CaCo-2 colon cancer cells. Moreover, both ligands inhibited c-Myc protein expression and its E-box DNA binding activity. Additionally, Mad1 protein expression and its E-box DNA binding activity were strongly increased by 15d-PG J2 and, to a lesser extent, by rosiglitazone. Sp1 transcription factor expression and its GC-box DNA binding activity were not affected by both PPARgamma ligands. Results obtained by transient transfection of CaCo-2 cells with pmaxFP-Green-PRL plasmid constructs containing the functional hTERT core promoter (including one E-box and five GC-boxes) and its E-box deleted sequences, cloned upstream of the green fluorescent protein reporter gene, demonstrated that 15d-PG J2, and with minor effectiveness, rosiglitazone, strongly reduced hTERT core promoter activity. E-boxes for Myc/Mad/Max binding showed a higher activity than GC-boxes for Sp1. By using GW9662, an antagonist of PPARgamma, we demonstrated that the effects of 15d-PG J2 are completely PPARgamma independent, whereas the effects of rosiglitazone on hTERT expression seem to be partially PPARgamma independent. The regulation of hTERT expression by 15d-PG J2 and rosiglitazone, through the modulation of the Myc/Max/Mad1 network, may represent a new mechanism of action of these substances in inhibiting cell proliferation.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Neoplasias del Colon/metabolismo , Prostaglandina D2/análogos & derivados , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Telomerasa/antagonistas & inhibidores , Tiazolidinedionas/farmacología , Western Blotting , Células CACO-2 , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , PPAR gamma/metabolismo , Regiones Promotoras Genéticas/genética , Prostaglandina D2/farmacología , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Reacytive Oxygen Species (ROS) have long been considered to be involved in the initiation, progression and metastasis of cancer. However, accumulating evidence points to the benefical role of ROS. Moreover, ROS production, leading to apoptosis, is the mechanism by which many chemotherapeutic agents can act. Beside direct actions, ROS elicit lipid peroxidation, leading to the production of 4-hydroxynoneal (HNE). Interestingly, HNE also seems to have a dual behaviour with respect to cancer. In this review we present recent literature data which outline the "two-faced" character of oxidative stress and lipid peroxidation in carcinogenesis and in the hallmarks of cancer.
RESUMEN
The effects of polyunsaturated fatty acids (PUFAs) obtained from the diet on colorectal cancer have been widely explored. However, controversial results have been obtained about the role played by the lipid peroxidation products of PUFAs, such as 4-hydroxy-nonenal (HNE), in the control of colon cancer growth. This aldehyde, indeed, showed both procarcinogenic and protective effects. In an attempt to verify the action of HNE, we studied the effects of a low dose of HNE (1 microM), similar to those "physiologically" found in normal cells and plasma, on telomerase activity, a key parameter of malignant transformation. Caco-2 cells were exposed to HNE and, paralleling cell growth inhibition, we observed the down-regulation of telomerase activity and hTERT expression. Similar effects have also been observed in HT-29 cells, in which HNE inhibited cell proliferation, telomerase activity and hTERT expression, suggesting that the inhibition of telomerase activity could be a general mechanism involved in the antiproliferative effect exerted by this aldehyde. Finally, we elucidated the mechanism of hTERT inhibition by HNE. A reduction of GSH content preceded the decrease of telomerase activity, but this only partially explained the telomerase activity inhibition. The major mechanism of HNE action seems to be the modulation of expression and activity of transcription factors belonging to the Myc/Mad/Max network. Since the presence of PUFAs in the diet exposes epithelial colon cells to HNE, this aldehyde could contribute to cell growth control through the inhibitory action on telomerase activity and hTERT expression, suggesting a protective effect on colon mucosa.
Asunto(s)
Aldehídos/farmacología , Telomerasa/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Regulación hacia Abajo , Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Células HT29 , Humanos , Peroxidación de Lípido/efectos de los fármacos , Telomerasa/biosíntesisRESUMEN
HNE (4-hydroxynonenal), the major product of lipoperoxidation, easily reacts with proteins through adduct formation between its three main functional groups and lysyl, histidyl and cysteinyl residues of proteins. HNE is considered to be an ultimate mediator of toxic effects elicited by oxidative stress. It can be detected in several patho-physiological conditions, in which it affects cellular processes by addition to functional proteins. We demonstrated in the present study, by MS and confirmed by immunoblotting experiments, the formation of HNE-alpha-enolase adduct(s) in HL-60 human leukaemic cells. Alpha-enolase is a multifunctional protein that acts as a glycolytic enzyme, transcription factor [MBP-1 (c-myc binding protein-1)] and plasminogen receptor. HNE did not affect alpha-enolase enzymatic activity, expression or intracellular localization, and did not change the expression and localization of MBP-1 either. Confocal and electronic microscopy results confirmed the plasma membrane, cytosolic and nuclear localization of alpha-enolase in HL-60 cells and demonstrated that HNE was colocalized with alpha-enolase at the surface of cells early after its addition. HNE caused a dose- and time-dependent reduction of the binding of plasminogen to alpha-enolase. As a consequence, HNE reduced adhesion of HL-60 cells to HUVECs (human umbilical vein endothelial cells). These results could suggest a new role for HNE in the control of tumour growth and invasion.
Asunto(s)
Aldehídos/administración & dosificación , Aductos de ADN/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Plasminógeno/metabolismo , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células HL-60 , HumanosRESUMEN
Telomerase enzyme, containing a catalytic subunit, the human telomerase reverse transcriptase (hTERT), and a small integral RNA component, synthesises the telomeres, the ends of eukaryotic chromosomes. Inhibition of telomerase activity leads the cells to senescence and death. Myelodysplastic syndromes (MSD) are hematological malignancies characterized by peripheral blood cytopenia and ineffective hematopoiesis. Telomerase activity and hTERT expression in MDS patients were independently investigated by different groups obtaining contradictory results. We analyzed telomerase activity and hTERT expression in the bone marrow of ten control, 15 MDS patients and two patients with AML, likely evolved from a previous MDS. Moreover, the expression of c-myc, mad1, p53 (transcription factors involved in hTERT expression regulation), has been investigated. Telomerase activity and hTERT expression increased in the MDS patients with respect to the controls. The analysis of the MDS subgroups, indicated that patients with more severe disease demonstrated significantly higher levels of hTERT expression and telomerase activity with respect to the patients with more favorable disease. c-Myc and p53 expressions were not significantly different between controls and MDS patients, whereas mad1 expression was increased in MDS patiens, particularly in those with more favorable disease. We hypothesize that mad1 increase can contribute to reduce the hTERT expression in the early stage of disease and we suggest that hTERT expression and telomerase activity, whether confirmed in larger series of cases could support other parameters in the diagnosis and stadiation of MDS.
Asunto(s)
Síndromes Mielodisplásicos/genética , Telomerasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/diagnóstico , Pronóstico , ARN Mensajero/análisis , Factores de Riesgo , Telomerasa/análisis , Telomerasa/metabolismo , Factores de Transcripción/genéticaRESUMEN
4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p<0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.
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Aldehídos/farmacología , Diferenciación Celular/efectos de los fármacos , Leucemia/tratamiento farmacológico , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Proliferación Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Células HL-60 , Humanos , Leucemia/patología , Peroxidación de Lípido/efectos de los fármacos , MicroARNs/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Activación TranscripcionalRESUMEN
The role of the Notch1 pathway has been well assessed in leukemia. Notch1 mutations are the most common ones in T acute lymphoblastic leukaemia patients which carry either oncogenic Notch1 forms or ineffective ubiquitin ligase implicated in Notch1 turnover. Abnormalities in the Notch1-Jagged1 system have been reported also in acute myelogenous leukaemia (AML) patients where Jagged1 is frequently over-expressed. Moreover, activating Notch1 mutations, as well, can occur in human AML and in leukemia cases with lineage infidelity. As a result, Notch1 signalling inhibition is an attractive goal in leukaemia therapy. Blockage/delay in cell differentiation and/or increase of proliferation are the main results of Notch1 signalling activation in several leukemic cell lines. Moreover, specific genes involved in cell growth control have been identified as Notch1 transcriptional targets, i.e. Cyclin D1 and c-Myc. 4-Hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, is involved in several pathological and physiological conditions, including inflammation; atherosclerosis; and neurodegenerative and chronic liver diseases. Moreover HNE has an antiproliferative/ differentiative effect in several cell lines, by affecting the expression of key genes, such as oncogenes (e.g. c-Myc, c-Myb), cyclins and telomerase. This prompted us to study the effect of HNE on Notch1 expression and its related signalling in HL-60 cells, a leukemic cell line widely used for differentiation studies. RT-PCR as well as Western blot assay showed Notch1down-regulation in HNE-treated HL-60 cells. The expression of Hes1, a Notch1 target gene, was concomitantly down-regulated by HNE treatment, reflecting Notch1 signalling inhibition. DAPT, an inhibitor of Notch activity, when added contemporary to HNE, further increased cell growth inhibition, without affecting apoptosis. Moreover, DAPT treatment reversed the HNE-induced differentiation. Overall these results suggest that Notch1 is a target for HNE and its down regulation is a key event in HNE-mediated inhibition of cell proliferation in the HL-60 cell line. By contrast our data do not support a role for Notch1 in HNE- induced differentiation or apoptosis.
Asunto(s)
Aldehídos/farmacología , Peroxidación de Lípido/efectos de los fármacos , Receptor Notch1/genética , Receptor Notch1/fisiología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Western Blotting , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Células HL-60 , Humanos , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triglicéridos/farmacología , Ácido gamma-Aminobutírico/análogos & derivados , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Peroxisome proliferators-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. The three PPAR isoforms (alpha, gamma and beta/delta) have been found to play a pleiotropic role in cell fat metabolism. Furthermore, in recent years, evidence has been found regarding the antiproliferative, proapoptotic, and differentiation-promoting activities displayed by PPAR ligands, particularly by PPARgamma ligands. PPAR ligands affect the expression of different growth-related genes through both PPAR-dependent and PPAR-independent mechanisms. Moreover, an interaction between PPAR ligands and other molecules which strengthen the effects of PPAR ligands has been described. Here we review the action of PPAR on the control of gene expression with particular regard to the effect of PPAR ligands on the expression of genes involved in the regulation of cell-cycle, differentiation, and apoptosis. Moreover, the interaction between PPAR ligands and 4-hydroxynonenal (HNE), the major product of the lipid peroxidation, has been reviewed.
RESUMEN
PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.
Asunto(s)
Aldehídos/farmacología , Neoplasias del Colon/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Prostaglandina D2/análogos & derivados , Tiazolidinedionas/farmacología , Apoptosis/genética , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Sinergismo Farmacológico , Expresión Génica/efectos de los fármacos , Humanos , Ligandos , PPAR gamma/metabolismo , Prostaglandina D2/farmacología , Rosiglitazona , Proteína X Asociada a bcl-2/genéticaRESUMEN
4-Hydroxynonenal (HNE), produced during oxidative stress, has an antiproliferative/differentiative effect in several tumor cells. Recently, it has been observed that oxidative stress accelerates telomere loss. The length of telomeres depends on the telomerase activity, and the catalytic subunit of telomerase (hTERT) is strongly up-regulated in most human cancers and inhibited by differentiating agents. In this paper the inhibitory effect of HNE on telomerase activity and hTERT expression in three human leukemic cell lines (HL-60, U937, ML-1) is reported. To investigate the molecular mechanism involved in hTERT down-regulation by HNE, the expression of several transcription factors was also studied: in all these cell lines, c-Myc was inhibited, Mad-1 was up-regulated, and Sp-1 was not affected. Moreover, in p53 wild-type ML-1 cells, HNE up-regulated p53 expression. In HL-60 cells, DNA binding activity of c-Myc and Mad-1 to the E-box sequence of the hTERT promoter was inhibited and up-regulated, respectively. In summary, HNE inhibits telomerase activity via decreased hTERT promoter activity, by modulating c-Myc/Mad-1 transcription factor expression.
Asunto(s)
Aldehídos/farmacología , Proteínas de Unión al ADN/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Telomerasa/efectos de los fármacos , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Estrés Oxidativo/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/biosíntesis , Telomerasa/genética , Telomerasa/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Lipid peroxidation is very low in proliferating cells and tumours and it might have a role in the regulation of cell proliferation and differentiation by acting through its products. 4-hydroxynonenal (HNE) has been proposed as a mediator of lipoperoxidation effects. It has been demonstrated that HNE can inhibit cell growth and induce differentiation in different leukemic cell lines. The onset of differentiation, induced by HNE, was accompanied by a reduction of c-myc expression. In HL-60 cells, HNE induced the accumulation of cells in the G0/G1 phase of the cell cycle. Cell cycle progression is regulated by three protein classes, the cyclins, the cyclin-dependent kinases (CDKs), and the CDK inhibitors (CKIs). In HL-60 cells, HNE decreased the expression of cyclin D1, D2 and A and caused an increase of p21 (the most important CKI) expression, whereas it did not affect CDK expressions. Since cyclins D/CDK2 and cyclin A/CDK2 phosphorylate pRB, HNE caused an increase of hypophosphorylated pRb. Hypophosphorylated pRb binds and inactivates the E2F transcription factors. Band-shift experiments demonstrated that HNE caused a decrease of "free" E2F, as well as an increase of pRb (and pRB family members) bound to E2F with consequent repression of the transcription.
Asunto(s)
Aldehídos , Ciclo Celular/fisiología , Aldehídos/farmacología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/efectos de los fármacos , Quinasas Ciclina-Dependientes/genética , Ciclinas/efectos de los fármacos , Ciclinas/genética , Expresión Génica/efectos de los fármacos , Genes myc/efectos de los fármacos , Células HL-60/citología , Humanos , Peroxidación de LípidoRESUMEN
4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.