RESUMEN
BACKGROUND: Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive. RESULTS: To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis. CONCLUSIONS: Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.
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Ciclina D1/metabolismo , Ciclinas/metabolismo , Mieloma Múltiple/metabolismo , Animales , Ciclo Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Separación Celular , Embrión de Pollo , Ciclina D1/genética , Ciclinas/genética , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Desnudos , Mieloma Múltiple/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The suppression of oestrogen receptor alpha (ERalpha) functions by silencing RNAs in association with or not with anti-oestrogens (AEs) both in vitro and in breast cancer cell xenografts was assessed. In vitro, a prolonged decrease in ERalpha protein expression and an enhanced AE-induced inhibition of ERalpha-mediated transcription, together with antiproliferative activity, were observed. Incorporation of ERalpha-siRNAs in pegylated nanocapsules (NC) was achieved; and their intravenous injections in MCF-7 xenografts, in contrast to scramble siRNA containing NCs, lead to decrease in ERalpha protein content and Ki67 labelling in tumour cells. The pure AE RU58668 (RU) both free and entrapped in stealth nanospheres (NS) at very low concentration (8 microg/kg/week) had no effect on tumour growth evolution. However, coinjection of the two nanocarriers potentiated the decrease in ERalpha protein, concomitantly with decreasing tumour vasculature and glucose transporter-1. These data support that the targeted delivery of ERalpha-siRNA in breast tumours potentiates the inhibition of E(2)-induced proliferative activity by encapsulated AE through enhanced anti-vascular activity. In the hormone-independent MDA-MB-231 xenograft model, RU-NS at 4 mg/kg/week induce also a strong tumour vascular normalisation. Together, these findings suggest that the anti-oestrogen activity of RU as well as that of targeted ERalpha-siRNA leads to anti-angiogenic activity. Their delivery in stealth nanocarriers may constitute a new anti-cancer therapeutic strategy in solid tumours.
Asunto(s)
Adenocarcinoma/patología , Neoplasias de la Mama/patología , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/uso terapéutico , Receptor alfa de Estrógeno/antagonistas & inhibidores , Estrógenos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Nanocápsulas/administración & dosificación , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias Hormono-Dependientes/patología , ARN Interferente Pequeño/uso terapéutico , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/trasplante , Sinergismo Farmacológico , Estradiol/farmacología , Estradiol/uso terapéutico , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inyecciones Intravenosas , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Nanocápsulas/química , Nanosferas/administración & dosificación , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Poliésteres , Polietilenglicoles , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Organismos Libres de Patógenos Específicos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To determine the better liposomal formulation incorporating the active metabolite of tamoxifen, 4-hydroxy-tamoxifen (4HT) and the biological impact of 4HT-pH-gradient liposomes on response to in vivo treatment. METHODS: Several pegylated liposomes were formulated by varying the composition of lipids, increasing external pH from 7.4 to 9.0 and doubling the lipid concentration. Dipalmitoylphosphatidylcholine / cholesterol / distearoylphosphoethanolamine poly(ethylene)glycol liposomes (DL-9 liposomes) were chosen for their physico-chemical properties. Toxicity and release kinetics were assessed in breast cancer MCF-7 as well as in multiple myeloma (MM) cells. In vivo antitumor activity and bio-distribution were measured in the RPMI8226 MM model. RESULTS: Compared to conventional non-pH-gradient liposomes, 4HT-DL-9 liposomes resulted in concentration of up to 1 mM 4HT, greater stability, relative toxicity and slow 4HT release. Intravenous injections of 4HT-DL-9 liposomes at 4 mg/kg/week blocked MM tumor growth. Ki67 and CD34 labeling decreased in treated tumors, concomitantly with increase of activated caspase-3 supporting a cell proliferation arrest, a decrease of tumor vasculature and the induction of tumor cell death. CONCLUSION: This antitumor effect was assumed to be the result of a modified biodistribution of 4HT once trapped in DL-9 liposomes. Such 4HT-containing pH-gradient Stealth nanocarriers could be helpful for MM treatment.
Asunto(s)
Modelos Animales de Enfermedad , Mieloma Múltiple/tratamiento farmacológico , Fuerza Protón-Motriz/efectos de los fármacos , Tamoxifeno/análogos & derivados , Animales , Línea Celular Tumoral , Femenino , Humanos , Liposomas , Ratones , Ratones Desnudos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Fuerza Protón-Motriz/fisiología , Tamoxifeno/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The cochaperone p23 is required for the chaperoning cycle of hsp90 and to enhance the maturation of several client proteins. Tosylcyclonovobiocic acids (4TCNA and 7TCNA) are potent analogs of novobiocin and induce cell cycle arrest, apoptosis and degradation of hsp90 client proteins in a panel of cancer cells. In this study, Western blotting shows that 4TCNA and 7TCNA triggered processing of the hsp90 cochaperone p23 in a dose-dependent manner. Small interfering RNA (siRNA)-mediated reduction of p23 expression in MCF-7 breast cancer cells did not block 4TCNA-induced caspase activation as assessed by the cleavage of PARP. This result indicates that 4TCNA-mediated cell death is a p23-independent process. In HT29 colon cancer cells, 4TCNA and 7TCNA up-regulated GRP78 and GRP94 supporting involvement of ER stress in apoptosis.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Oxidorreductasas Intramoleculares/efectos de los fármacos , Neoplasias/enzimología , Novobiocina/análogos & derivados , Novobiocina/farmacología , Apoptosis , Línea Celular Tumoral , Colágeno Tipo XI/metabolismo , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Receptor alfa de Estrógeno/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Prostaglandina-E Sintasas , Interferencia de ARN , ARN Interferente Pequeño/genética , Estrés Fisiológico , Regulación hacia ArribaRESUMEN
Multiple myeloma (MM) is a malignancy characterized by the accumulation of tumoral plasma cells in bone marrow. This disease remains incurable and the development of new therapeutic strategies is urgently required. We have studied the effects of 2 selective estrogen receptor disrupters (SERDs), RU 58668 (RU) and ICI 182,780 (ICI) or pure antiestrogens (AEs) on MM cell lines. Both compounds have antimyeloma activity through either cell cycle arrest or induction of apoptosis. To analyze the molecular mechanisms of SERD action, we choose 2 differently responding cell lines as models. In LP-1 cells, RU blocked cell cycle at the G1 phase. RU treatment induced a rapid decrease of c-Myc, an upregulation of p27(Kip1), and the subsequent decreased activity of cyclin-dependent kinase, CDK6 and associated cyclin D3, impairing the inactivation of the retinoblastoma protein (pRb). In RPMI 8226 cells, RU induced apoptosis by recruiting endoplasmic reticulum- as well as mitochondria-associated caspases. Moreover, RU interfered with the NF-kappaB survival pathway, often deregulated in MM malignancy. Antimyeloma activities were observed in dexamethasone (Dex)- and RU-resistant cells when RU was combined with bortezomib; Dex and bortezomib being frequently used in MM therapy. RU induced the death of CD138+ cells purified from MM patients but not CD19+ normal cells obtained from tonsils. Therefore, RU mediates the inhibition of survival, the activation of apoptosis and finally potentiates anticancer drug. Those combinatory effects provide a basis for the potential use of pure AEs in MM treatment.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Caspasas/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Retículo Endoplásmico/metabolismo , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/farmacología , Fase G1/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Pirazinas/farmacología , Western Blotting , Bortezomib , Proliferación Celular/efectos de los fármacos , Colorimetría , Ciclina D3 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Estradiol/farmacología , Citometría de Flujo , Humanos , Inmunoprecipitación , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Specific siRNAs that target estrogen receptor alpha (ERalpha) were encapsulated in nanocapsules (NCs). We produced small (approximately 100-200 nm) ERalpha-siRNA NCs with a water core by incorporating two mixed duplexes of specific ERalpha-siRNAs (ERalpha-mix-siRNA) into NCs. The encapsulation yield that was obtained with poly(iso-butylcyanoacrylate) (PIBCA) NCs was low, whereas no release of trapped siRNA was observed for poly(ethylene)glycol-poly(D,L-lactide-co-glycolide) (PEG-PLGA) NCs. High levels of ERalpha-siRNA incorporation into PEG-epsilon-caprolactone-malic acid (PEG-PCL/MA) NCs (3.3 microM in a polymer solution at 16 mg/mL) were observed (72% yield). No difference in size or zeta potential was observed between siRNA NCs that were based on PEG-PCL/MA and empty NCs. Fluorescence quenching assays confirmed the incorporation of siRNA into the NC core. A persistent loss of ERalpha (90% over 5 days) was observed in MCF-7 human breast cancer cells that were exposed to PEG-PCL/MA NCs that were loaded with ERalpha-siRNA. The intravenous injection of these NCs into estradiol-stimulated MCF-7 cell xenografts led to a significant decrease in tumor growth and a decrease in ERalpha expression in tumor cells. These data indicate that a novel strategy, based on ERalpha-siRNA delivery, could be developed for the treatment of hormone-dependent breast cancers.
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Materiales Biocompatibles/química , Química Física/métodos , Receptor alfa de Estrógeno/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Emulsiones , Femenino , Humanos , Ensayo de Materiales , Ratones , Ratones Desnudos , Nanocápsulas/química , Trasplante de NeoplasiasRESUMEN
A new series of coumarin inhibitors of hsp90 lacking the noviose moiety as well as substituents on C-7 and C-8 positions of the aromatic ring was synthesised and their hsp90 inhibitory activity has been delineated: for example, their capacity to induce the degradation of client proteins and to inhibit estradiol-induced transcription in human breast cancer cells. In cell proliferation assay, the most active compound 5g was approximately 8 times more potent than the parent novobiocin natural compound.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Cumarinas/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Novobiocina/farmacología , Antibióticos Antineoplásicos/síntesis química , Sitios de Unión , Neoplasias de la Mama/patología , Línea Celular Tumoral/efectos de los fármacos , Cumarinas/química , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Estradiol/farmacología , Humanos , Novobiocina/síntesis química , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacosRESUMEN
For the first time, two organometallic triphenylethylene compounds (Fc-diOH and DFO), with strong antiproliferative activity in breast cancer cells, but insoluble in biological fluids, were incorporated in two types of stealth nanoparticles (NP): PEG/PLA nanospheres (NS) and nanocapsules (NC). Their physicochemical parameters were measured (size, zeta potential, encapsulation and loading efficiency), and their biological activity was assessed. In vitro drug release after high dilution of loaded NPs was measured by estradiol binding competition in MELN cells. The influence of the encapsulated drugs on the cell cycle and apoptosis was studied by flow cytometry analyses. Notwithstanding potential drug adsorption at the NP surface, Fc-diOH and DFO were incorporated efficiently in NC and NS, which slowly released both compounds. They arrested the cell cycle in the S-phase and induced apoptosis, whose activity is increased by loaded NS. A decrease in their antiproliferative activity by the antioxidant alpha-tocopherol indicated that reactive oxygen species (ROS) may be involved. Therefore, nanosystems, containing for the first time a high load of anticancer organometallic triphenylethylenes, have been developed. Their small size and delayed drug release, combined with their enhanced apoptotic potential, are compatible with an increased persistence in the blood and a promising antitumour activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Ferrosos/química , Nanopartículas/química , Tamoxifeno/farmacología , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Estradiol/química , Estradiol/farmacología , Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Metalocenos , Estructura Molecular , Nanocápsulas/química , Nanosferas/química , Tamaño de la Partícula , Receptores de Estrógenos/antagonistas & inhibidores , Electricidad Estática , Propiedades de Superficie , Tamoxifeno/química , alfa-Tocoferol/química , alfa-Tocoferol/farmacologíaRESUMEN
Multiple myeloma (MM) is a common hematological malignancy which remains incurable due to both intrinsic and acquired resistance to conventional or more novel drugs. Estrogenic and antiestrogenic compounds are very promising drugs for the treatment of MM. Indeed, they inhibit cell proliferation in vitro. They block cell cycle and/or induce apoptosis even in drug-resistant MM cells but not normal B cells. They interfere with survival pathways often deregulated in myelomas. They co-operate with conventional drugs to enhance apoptosis or to overcome resistance. In vivo, they act also on tumoral angiogenesis in xenograft models. As a whole, they possess all the criteria which render them attractive for a new therapeutic strategy. Importantly, they are well-tolerated at the doses tested in vitro or in vivo, encouraging the rapid onset of critical trials.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Receptores de Estrógenos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/metabolismo , Humanos , Estructura Molecular , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Neovascularización Patológica/tratamiento farmacológico , Plasmacitoma/tratamiento farmacológico , Plasmacitoma/metabolismo , Plasmacitoma/patología , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de SeñalRESUMEN
Selective hsp90 inhibitors simultaneously destabilize and deplete key signaling proteins involved in cell proliferation and survival, angiogenesis, and metastasis. Investigation of novobiocin analogues lacking the noviose moiety as novel inhibitors of hsp90 was carried out. A novel series of 3-aminocoumarin analogues has been produced and screened in cell proliferation, and the molecular signature of hsp90 inhibition was assessed by depletion of estrogen receptor, HER2, Raf-1, and cdk4 in human breast cancer cells. This structure-activity relationship study highlights the crucial role of the C-4 and/or C-7 positions of coumarin which appeared to be essential for degradation of hsp90 client proteins. Removal of the noviose moiety in novobiocin together with introduction of a tosyl substituent at C-4 or C-7 coumarins provides 6e and 6f as lead structures which compared favorably with novobiocin as demonstrated by enhanced rates of cell death. The processing and activation of caspases 7 and 8 and the subsequent cleavage of PARP by 6e suggest stimulation of the extrinsic apoptosis pathway.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Novobiocina/análogos & derivados , Novobiocina/síntesis química , Antineoplásicos/farmacología , Apoptosis , Neoplasias de la Mama , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Citometría de Flujo , Humanos , Novobiocina/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacosRESUMEN
Oestrogen receptors (ER)alpha and beta modify the expression of genes involved in cell growth, proliferation and differentiation through binding to oestrogen response elements (EREs) located in a number of gene promoters. Transient transfection of different luciferase reporter vectors 3xEREs-Vit, 2xEREs-tk and ERE-C3 showed that the transactivation capacity of both ER subtypes was influenced by 1) the nature of the inducer (oestradiol (E2), phyto- and anti-oestrogen (AE)), 2) the structure of the promoter (nucleotidic sequence, number of ERE, length of the promoter sequence) and 3) the cell line (containing endogenous ER (MCF-7) or in which ER was stably expressed (MDA-MB-231-HE-5 (ERalpha+) or MDA-MB-231-HERB (ERbeta+)). ER subtype did not display the same efficacy on the different constructions in the presence of E2 and of AE according to the cell (e.g. in MCF-7 cells: tk>>Vit>>C3 approximately 0 while in MDA-MB-231 cells: Vit>>tk approximately C3). E2 response was higher in MCF-7 cells, probably due to higher ER expression level (maximal at 10(-10)M instead of 10(-8)M for E2 in HE-5 cells). Finally, the same ligand could exert opposite activities on the same promoter according to the ER isoform expressed: in the MDA-MB-231 cells, AE acted as inducers of the C3 promoter via ERbeta whereas ERalpha/AE complexes down-regulated this promoter. Approximately 70% of breast tumours express ER and most tumour cells coexpress both ER isotypes. Thus, different types of ER dimers can be formed in such tumours (ERbeta or ERalpha homodimers or ERalpha/ERbeta heterodimers). We therefore studied the influence of the coexistence of the two ERs on the ligand-induced transcriptional process following transient transfection of ERalpha in ERbeta+ cells, and inversely ERbeta in ERalpha+ cells. ERbeta-transfection inhibited the E2- and genistein-induced ERalpha-dependent transcription on all promoters in all cell lines except C3 in MCF-7; this inhibitory effect was lost following transfection of ERbeta deleted of its AF-1 (ERbeta-AF-2). These results suggest that the dominant negative properties of ERbeta are mainly due to its AF-1 function. Interestingly, transfection of an ERbeta-AF-2 construct into MCF-7 cells potentiated the transcription inhibitory capacity of 4-OH-tamoxifen (OHT) on the Vit and tk promoters. Thus, (1) OHT exerts an agonistic activity through the AF-1 function of ER and (2) expression of ERbeta in breast cancer cells seems to favour the AE treatment. Contrary to ERbeta, ERalpha-transfection had little effect on ERbeta transactivation capacity in HERB cells. Finally, the ratio ERalpha/ERbeta constitutes one decisive parameters to orientate the transcriptional mechanism of a target gene in the presence of agonist as well as of antagonist ligands.
Asunto(s)
Estradiol/farmacología , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/química , Receptor beta de Estrógeno/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Elementos de Respuesta , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Genisteína/farmacología , Humanos , Proteínas Mutantes/genética , Isoformas de Proteínas , Estructura Terciaria de Proteína , Transducción de Señal/genética , Transcripción Genética/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
The folate receptor (FR) is a highly selective tumor marker over expressed in many human cancers and it constitutes a useful target for tumor-specific drug delivery. Thus, the conjugation of folic acid to different drugs or drug carriers may enhance the delivery of the therapeutic agent to FR-positive tumor cells. The aim of this study was to investigate the interactions of folate-conjugated polyalkylcyanoacrylate nanoparticles with tumor cells overexpressing the FR. For this purpose, nanoparticles were prepared by nanoprecipitation of the poly[aminopoly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H2NPEGCA-co-HDCA)] copolymer and labeled with the hydrophobic fluorescent dye nile red. Nile red-loaded nanoparticles were then conjugated to folic acid via the PEG terminal amino groups. Four human cancer cell lines were then tested by western blot in order to evaluate the FR expression levels. KB3-1 cell line showed the higher expression level, while MCF-7 cells were taken as a control. After measuring the cytotoxicity of the nanoparticles on these two cell lines, fluorescent folate-nanoparticles were incubated with them and the cellular uptake was evaluated by confocal microscopy and flow cytometry. KB3-1 cells showed a greater nanoparticle internalization, when compared to MCF-7 cells.
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Cianoacrilatos/química , Ácido Fólico/química , Modelos Biológicos , Nanopartículas , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Humanos , Microscopía Confocal , Microscopía FluorescenteRESUMEN
The aim of this study was to develop both a physical and a chemical protection of the anticancer drug gemcitabine, which suffers from a rapid plasmatic metabolization. For this purpose, we used a series of lipophilic derivatives of gemcitabine in which an acyl chain is covalently coupled to the 4-amino group of gemcitabine; moreover, a physical protection of the drug was attempted by incorporating these lipophilic derivatives into poly(H(2)NPEGCA-co-HDCA) nanospheres and nanocapsules. Nanoparticles were prepared by nanoprecipitation of the poly(H(2)NPEGCA-co-HDCA) copolymer and their size, zeta potential and encapsulation efficiency were further characterized. These results have been relied on lipophilicity and flexibility studies. Data showed that only the more lipophilic derivative, 4-(N)-stearoylgemcitabine, was incorporated with a high yield. Thus, 4-(N)-stearoylgemcitabine-containing nanospheres and nanocapsules were further analyzed by differential scanning calorimetry. Their cytotoxicity was tested on two human cancer cell lines and compared to that of gemcitabine and free 4-(N)-stearoylgemcitabine.
Asunto(s)
Antimetabolitos Antineoplásicos/química , Cianoacrilatos , Desoxicitidina/análogos & derivados , Nanocápsulas , Nanosferas , Antimetabolitos Antineoplásicos/farmacología , Rastreo Diferencial de Calorimetría , Línea Celular Tumoral , Desoxicitidina/química , Desoxicitidina/farmacología , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Humanos , Polímeros , Profármacos/química , Relación Estructura-Actividad , GemcitabinaRESUMEN
Estrogens are essential for human health. Their physiological effects are primarily mediated by two types of intracellular estrogen receptors (ER alpha and ER beta) that function as DNA-binding transcription factors. However, estrogens are also involved in the development and progression of breast cancers. Endocrine therapy aims to reduce the availability of the hormone or to counteract its action. This can be achieved by preventing estrogen production or administrating antiestrogens (AEs), synthetic drugs belonging to several distinct structural categories. Selective estrogen receptor modulators (or SERMs) bind ERs but have a mixed agonist/antagonist profile. Selective estrogen receptor downregulators (or SERDs) are pure antiestrogens, acting by decreasing the level of ERs through their ubiquitinylation and subsequent targeting to the proteasome. We review most of the usual antiestrogenic therapies for estrogen-dependent and estrogen-independent solid cancers and present our recent results on the use of AEs against multiple myeloma. In breast cancer treatments, the most commonly used antiestrogen, tamoxifen, has major limitations: side effects due to its partial agonist activity and constitutive or acquired resistance. We propose that novel AE drug delivery systems may enhance the overall beneficial effects by targeting tumoral cells.
Asunto(s)
Antagonistas de Estrógenos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Animales , Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/metabolismo , Antagonistas de Estrógenos/farmacología , Humanos , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismoRESUMEN
Anti-oestrogens (AEs) are currently used for treating hormone-dependent breast cancers. They specifically bind to oestrogen receptors (ERs) and inhibit their transactivation capacity. However, ERs are present in various other tissues in which AEs may have either a beneficial or detrimental action. AE administration via systems targeting breast tumours may be an important therapeutic improvement. Thus, several biodegradable drug delivery systems containing either "mixed" (4-hydroxytamoxifen - 4-HT) or "pure" (RU 58668 - RU) AEs were prepared. Liposomes and nanospheres (NS, composed of non-toxic and biodegradable lipids and poly(d,l-lactic acid) incorporated up to 1 and 0.5 mM AE, respectively. Nanocapsules (NCs) in which an oily core solubilises the AE incorporated no more than 0.02 mM of the drug. PEG-functionalised nanoparticles survived longer in plasma and had better controlled release of the drug. The small size of the vectors (100-250 nm) was compatible with their extravasation through the discontinuous endothelium of tumour vasculature, allowing their accumulation in MCF-7 cell xenografts and leading to a prolonged exposure of the tumour to AEs. In these tumours and in MCF-7/ras xenografts, RU-NS and RU-NC (6.5mg/kg/week and 0.27 mg/kg/week, respectively, doses at which free RU had a very weak effect), both inhibited tumour growth. Entrapped RU significantly induced involution of tumours and strongly induced apoptosis in tumour cells, concomitantly with inhibiting tumour angiogenesis. 4-HT-nanoparticles also arrest oestradiol-induced tumour growth, inducing apoptosis and inhibiting angiogenesis. However, unlike RU-nanoparticles, they did not promote ERalpha subtype loss in tumour cells. Subcutaneous administration of both RU- and 4-HT-NS in MCF-7 xenografts strongly arrested tumour growth for prolonged periods and RUNS decreased the number of tumour epithelial cells. Analysis of the proteins involved in cell cycle proliferation and apoptosis confirmed that RU-nanoparticles were more efficient than 4-HT-nanoparticles. Their lack of toxicity and high anti-tumour potency that affects only tumour cells in the xenograft models mean these AE-loaded colloidal systems are a breakthrough in hormone-dependent breast cancer treatment.
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Coloides , Sistemas de Liberación de Medicamentos , Moduladores de los Receptores de Estrógeno/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Nanocápsulas , Receptores de Estrógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Nanopartículas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In most of multiple myeloma (MM) cells, the "pure" antiestrogen (AE) RU 58668 (RU) induced either a G1-arrest (LP-1, OPM-2, NCI-H929, U266 cells) or apoptosis (RPMI 8226 cells). In RPMI 8226 cells, RU activates a caspase-dependent cell death pathway leading to the release of cytochrome c, the decrease of the essential MM survival factor Mcl-1, the cleavage of Bid and the activation of caspases-3 and -8. Incorporation of RU in pegylated cholesterol-containing liposomes allowed a controlled RU release, improving its anti-proliferative and apoptotic effects in cells. In RPMI 8226 xenografts, i.v. injected RU-liposomes but not free RU, exhibited antitumor activity. In vivo, RU-liposomes triggered the mitochondrial death pathway, concomitantly with a down-regulation of Mcl-1 and Bid cleavage. The decrease of CD34 immunoreactivity indicated a reduction of angiogenesis. The decrease of VEGF secretion in vitro supported a direct effect of RU on angiogenesis. These pro-apoptotic and antiangiogenic effects were explained by a prolonged exposure to the drug and to the endocytosis capacity of liposomes which might increase RU uptake and bypass a membrane export of free RU. Thus, these combined enhanced activities of RU-liposomes support that such a delivery of an AE may constitute a strategy of benefit for MM treatment.
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Antineoplásicos/farmacología , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Liposomas , Mieloma Múltiple/tratamiento farmacológico , Animales , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Preparaciones de Acción Retardada , Sistemas de Liberación de Medicamentos/métodos , Estradiol/aislamiento & purificación , Estradiol/farmacología , Antagonistas de Estrógenos/aislamiento & purificación , Moduladores de los Receptores de Estrógeno/administración & dosificación , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Modelos Biológicos , Mieloma Múltiple/patología , Tonsila Palatina/citología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Multiple myeloma is an incurable B-cell malignancy requiring new therapeutic strategies. Our approach was to analyze the in vitro effects of a selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT), on six multiple myeloma cell lines. EXPERIMENTAL DESIGN: Cultured multiple myeloma cells were treated with various 4-OHT concentrations and the cellular response was studied: cell proliferation, cell viability, induction of apoptosis, caspase activities, and expression of signaling proteins. RESULTS: We found that pharmacologic concentrations of 4-OHT inhibit cell proliferation (4 of 6 cell lines). This inhibition is achieved by two independent events: a block at the G(1) phase of the cell cycle and the induction of apoptotic death. The cellular response to 4-OHT depends on the presence of functional estrogen receptors. 4-OHT treatment activates an intrinsic mitochondrial caspase-9-dependent pathway but not the Fas/FasL death pathway. Signaling pathways known to be involved in the survival and/or proliferation of multiple myeloma cells are not affected by 4-OHT treatment. 4-OHT-induced G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitor p27(Kip1) and the down-regulation of c-Myc. Among the Bcl-2 family members tested, the proapoptotic BimS protein is induced whereas the antiapoptotic protein Mcl-1 is decreased. CONCLUSIONS: Although the effects of 4-OHT are observed at micromolar concentrations, cellular mechanisms responsible for G(1) arrest, as well as apoptosis induction, are similar to those observed in breast cancer cells. Our data support the concept that 4-OHT may represent an alternative approach to inhibit proliferation and induce apoptosis of multiple myeloma cells.
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Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Proteínas Supresoras de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 9 , Caspasas/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación hacia Abajo , Fase G1/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mieloma Múltiple/patología , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/metabolismo , Células Tumorales CultivadasRESUMEN
Tamoxifen, a selective estrogen-receptor modulator, is effective in the treatment and prevention of breast cancer, but therapeutic resistance is common. Pure steroidal antiestrogens are efficacious in tamoxifen-resistant disease and, unlike tamoxifen, arrest cells in a state of quiescence from which they cannot reenter the cell cycle after growth factor stimulation. We now show that in hydroxytamoxifen-treated cells, transduction of the cell cycle inhibitor p27(Kip1) induces quiescence and insensitivity to growth stimulation by insulin/insulin-like growth factor I and epidermal growth factor/transforming growth factor alpha. Furthermore, reinitiation of cell cycle progression by insulin/insulin-like growth factor I in hydroxytamoxifen-arrested cells involves dissociation of the corepressors nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptor (SMRT) from nuclear estrogen receptor alpha and redistribution to the cytoplasm, a process that is inhibited by mitogen-activated protein/extracellular signal-regulated kinase, but not phosphatidylinositol 3'-kinase, inhibitors. These data suggest that agents that up-regulate p27(Kip1) or inhibit growth factor signaling via the extracellular signal-regulated kinases should be tested as therapeutic strategies in tamoxifen-resistant breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/fisiología , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/farmacología , Sustancias de Crecimiento/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Proteínas Supresoras de Tumor/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , División Celular/efectos de los fármacos , División Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno , Fulvestrant , Humanos , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Receptores de Estrógenos/metabolismo , Proteínas Represoras/metabolismo , Tamoxifeno/análogos & derivados , Transducción Genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genéticaRESUMEN
In MCF-7 (estrogen receptor (ER)+) and in MDA-MB-231 (ER-) cells stably transfected with either estrogen receptor alpha (ERalpha) or beta (ERbeta) subtype (MDA-MB-231 stably transfected with the mouse ERalpha cDNA (MERA) and MDA-MB-231 stably transfected with the human ERbeta cDNA (HERB), respectively) N-term heat shock protein of 90kDa (hsp90) ligands (geldanamycin and radicicol) and C-term hsp90 ligands (novobiocin) decrease the basal and estradiol (E(2))-induced transcription activity of ER on an estrogen responsive element (ERE)-LUC reporter construct concomitantly with or 1h after E(2) treatment. All hsp90 ligands induced an E(2)- and MG132-inhibited decrease of both ER cell content. However, the kinetics of these degradations are slower than those induced by the selective estrogen receptor down-regulator RU 58668 (RU). This suggests that inhibition of the hsp90 ATPase activity targets both ERs to the 26S proteasome and that hsp90 interacts with both ER subtypes. Rapamycin (Rapa) and cyclosporin A (CsA), ligands of immunophilins FK506 binding protein (FKBP52) and cyclophilin of 40kDa (CYP40) interacting in separate ER-hsp90 complexes, both induced a proteasomal-mediated degradation of ERs but not of their cognate immunophilin. Moreover, they also decrease the E(2)-induced luciferase transcription but weaker than RU and hsp90 ligands. Fluorescence activated cell sorter (FACS) analysis revealed a blockade of cell progression by RU and 4-hydroxy-tamoxifen at the G(1) phase of the cell cycle and an induction of apoptosis in MCF-7 cells. Rapa and mainly CsA (but not FK506) and hsp90 ligands promote by their own apoptosis in MCF-7, in MERA, and in HERB cells and in MDA-MB-231 ER-null cells. These data suggest that (1) hsp90, as for all steroid receptors, acts as a molecular chaperone for ERbeta; (2) ER-ligands (except tamoxifen), hsp90- and immunophilin-ligands (except FK506) target the two ER subtypes to a proteasome-mediated proteolysis via different signalling pathways; (3) hsp90- and immunophilin-ligands Rapa and CsA, alone or in association with anti-estrogens such as RU, may constitute a potential therapeutic strategy for breast cancer treatment.
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Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Inmunofilinas/farmacología , Neoplasias de la Mama , Línea Celular Tumoral , ADN Complementario/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Proteínas HSP90 de Choque Térmico/genética , Humanos , Cinética , Ligandos , Transcripción Genética/efectos de los fármacos , Activación Transcripcional , TransfecciónRESUMEN
Anticancer drug efficiency is governed by its bioavailability. In order to increase this parameter, we synthesized several injectable and biodegradable systems based on incorporation of anti-estrogens (AEs) in nanoparticles (NPs) and liposomes were synthesized. Both nanospheres (NS) and nanocapsules (NCs, polymers with an oily core in which AEs were solubilized) incorporated high amounts of 4-hydroxy-tamoxifen (4-HT) or RU 58668 (RU). Physico-chemical and biological parameters of these delivery systems, and coupling of polyethylene-glycol chains on the NP surface revealed to enhance the anti-tumoral activity of trapped AEs in a breast cancer MCF-7 cell xenograft model and to induce apoptosis. These features correlated with an augmentation of p21(Waf-1/Cip1) and of p27(Kip1) and a concomitant decrease of cyclin D1 and E in tumor extracts. Liposomes containing various ratios of lipids enhanced the apoptotic activity of RU in several multiple myeloma (MM) cell lines tested by flow cytometry. MM cell lines expressed both estrogen receptor alpha and beta subtypes except Karpas 620. Karpas 620 cells which did not respond to AEs became responsive following ER cDNA transfection. A new MM xenograft model was generated after s.c. injection of RPMI 8226 cells in nude mice. RU-loaded liposomes, administered i.v. in this model, at a dose of 12mgRU/kg/week, induced the arrest of tumor growth contrary to free RU or to empty liposomes. Thus, the drug delivery of anti-estrogens enhances their ability to arrest the growth of tumors which express estrogen receptors and are of particular interest for estrogen-dependent breast cancer treatment. In addition it represents a new potent therapeutic approach for multiple myeloma.