RESUMEN
Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30-40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5-6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.
Asunto(s)
Pollos , Daño del ADN , Animales , Carcinógenos , Ensayo Cometa , Femenino , Humanos , Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Roedores , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data. METHODS: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats. RESULTS: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats. CONCLUSIONS: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Asteraceae/química , Lactonas/farmacología , Sesquiterpenos/farmacología , Animales , Selectina E/metabolismo , Edema/tratamiento farmacológico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Interleucina-8/metabolismo , Masculino , Estructura Molecular , Monocitos/efectos de los fármacos , Componentes Aéreos de las Plantas/química , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
BACKGROUND: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance. METHODS: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated. RESULTS: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations. CONCLUSION: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 6 Dependiente de la Ciclina/genética , Resistencia a Antineoplásicos/genética , Estradiol/análogos & derivados , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/genética , Quimioterapia Adyuvante , Proteínas Cromosómicas no Histona/metabolismo , Estradiol/uso terapéutico , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/metabolismo , Humanos , Metiltransferasas/metabolismo , Piperazinas/uso terapéutico , Piridinas/uso terapéutico , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.
Asunto(s)
Acetohexamida/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Endotelio Linfático/efectos de los fármacos , Isoxsuprina/farmacología , Vasos Linfáticos/efectos de los fármacos , Nifedipino/farmacología , Proadifeno/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular , Quimiotaxis/efectos de los fármacos , Técnicas de Cocultivo , Sinergismo Farmacológico , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Hipoglucemiantes/farmacología , Metástasis Linfática , Vasos Linfáticos/irrigación sanguínea , Vasos Linfáticos/patología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/metabolismo , Células Tumorales Cultivadas , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics. METHODS: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs. RESULTS: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively. CONCLUSION: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Adhesión Celular/efectos de los fármacos , Endotelio Linfático/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/química , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Esferoides Celulares/efectos de los fármacos , Sulfonas/farmacología , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Quimiotaxis/efectos de los fármacos , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND: The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts. METHODS: To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs. RESULTS: We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with â¼50% attenuation of CCID formation by treatment of cells with 10 µM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general. INTERPRETATION: These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Neoplasias de la Mama/patología , Carcinoma/patología , Transdiferenciación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , FN-kappa B/fisiología , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Endoteliales/fisiología , Femenino , Humanos , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Invasividad Neoplásica , Nitrilos/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Células Tumorales CultivadasRESUMEN
Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon cancer model, using four sub-clones characterised by increasing FU-resistance derived from the cell line SW620. The resistance phenotype was preserved after FU withdrawal for 15weeks (~100 cell divisions) independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133(+) CD44(-) phenotype, and further exhibited loss of membrane-bound E-cadherin together with predominant nuclear ß-catenin localisation. Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple cellular strategies (epithelial-mesenchymal transition and selective propagation of CD133(+) cells). These resistance phenomena, in turn, accentuate the malignant phenotype.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Antígeno AC133 , Antígenos CD/metabolismo , Azacitidina/farmacología , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/patología , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Concentración 50 Inhibidora , Cinética , Mesodermo/efectos de los fármacos , Mesodermo/patología , Péptidos/metabolismo , Factores de Tiempo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups. METHODS: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by (14)C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers. RESULTS: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21(Cip/Waf) and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread. CONCLUSION: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ácido Gálico/análogos & derivados , Células HL-60/efectos de los fármacos , Estilbenos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Colorantes , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Ácido Gálico/farmacología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , Células HL-60/citología , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Transducción de Señal/efectos de los fármacosAsunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Fluorouracilo/uso terapéutico , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Reparación del ADN , Replicación del ADN , Relación Dosis-Respuesta a Droga , Epigénesis Genética , Femenino , Fluorouracilo/metabolismo , Fluorouracilo/farmacología , Expresión Génica , Genes p53/genética , Humanos , Metilación , Mitosis , Receptor ErbB-2/genética , Transducción de Señal/efectos de los fármacos , Uracil-ADN GlicosidasaRESUMEN
TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.
Asunto(s)
Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Proteína Letal Asociada a bcl/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Cromatina/metabolismo , Fragmentación del ADN/fisiología , ADN de Neoplasias/metabolismo , Embrión de Mamíferos/citología , Células HeLa/citología , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Ratones , Modelos Biológicos , Mutación , Proteína Oncogénica v-akt/metabolismo , Proteínas Quinasas/metabolismo , Ratas , Serina-Treonina Quinasas TOR , Transfección/métodos , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteína Letal Asociada a bcl/genéticaRESUMEN
Chemoresistance is a biological response of cells to survive toxic stress. During cancer treatment the development of chemoresistance is a major problem. The mechanisms how cells become insensitive, and which downstream pathways are affected are not completely understood. Since it has not been well analysed which and how many regulative disorders are subsummised under the term "chemoresistance", we examined and measured arabinosylcytosine (AraC)-mediated desensitation of two mechanisms relevant for tissue homeostasis, cell cycle inhibition and apoptosis induction. MCF-7 cells harbouring ectopic mutated p53 were suitable for this investigation because they activated these mechanisms subsequently and became insensitive to AraC with regard to cell cycle inhibition and apoptosis induction. The major causal mechanism of acquired resistance against AraC was most likely through the inhibition of the first step of AraC phosphorylation within the cell, which is rate limiting for its activation. With regard to cell cycle inhibition AraC-resistant cells were also resistant against 5-fluorodeoxyuridine (5-FdUrd), but fully responsive to 5-FdUrd-induced apoptosis, evidencing that cell cycle and apoptosis are independent of each other. Apoptosis correlated with AIF-activation and was independent of Caspase 7, whereas cell cycle inhibition correlated with cyclinD1 expression but not with induction of p21 or p27. The phosphate conjugated 5-FdUrd-araC heterodimer (5-Fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine), which is a prodrug of AraC-monophosphate, reactivated AIF and down-regulated cyclin D1 in AraC-resistant cells and circumvented resistance to apoptosis and to cell cycle inhibition. Also, cells which were resistant to 5-FdUrd or doxorubicin were sensitive to 5-FdUrd-araC. This investigation demonstrates that chemoresistance affects apoptosis induction and cell cycle inhibition independently and that detailed knowledge about the affected downstream pathways would enable the design of targeted intervention with small molecules to restore chemosensitivity.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Citarabina/farmacología , Resistencia a Antineoplásicos/fisiología , Floxuridina/farmacología , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular Tumoral , Citarabina/química , Citarabina/metabolismo , Femenino , Floxuridina/química , Floxuridina/metabolismo , Humanos , Estructura MolecularRESUMEN
In search for possible alternatives in the treatment of human malignancies we investigated several new heterodinucleoside phosphates consisting of 5-Fluorodeoxyuridine (5-FdUrd) and Arabinofuranosylcytosine (Ara-C). We show that all dimers tested inhibited the number of colonies of CCL228, CCL227, 5-FU resistant CCL227 and HT-29 human colon tumor cells with IC50 values ranging from 0.65 to 1 nM. Dimer # 2 inhibited the number of sensitive and Ara-C resistant H9 human lymphoma cells with IC50 values ranging from 200 to 230 nM. Since no significant difference in the cytotoxicity of the dimers could be observed between sensitive and resistant cells, these compounds might be used in the treatment of 5-FU and Ara-C resistant tumors.
Asunto(s)
Apoptosis , Citarabina/farmacología , Fosfatos de Dinucleósidos/química , Fluorouracilo/farmacología , Antimetabolitos Antineoplásicos/farmacología , Bisbenzimidazol/farmacología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Colorantes/farmacología , Dimerización , Colorantes Fluorescentes/farmacología , Humanos , Concentración 50 Inhibidora , Linfoma/tratamiento farmacológico , Propidio/farmacologíaRESUMEN
Cell division cycle 25A (Cdc25A) was shown to colocalise both with nuclear and cytoplasmic proteins. Recently, we have demonstrated that overexpressed Cdc25A promoted the survival of rat 423 cells through indirect activation of PKB-protein kinase B. Using a Cdc25A:ER fusion protein, which can be shuttled from the cytoplasm into the nucleus, the present investigation evidences that the antiapoptotic effect of Cdc25A was restricted to its cytoplasmic localisation in rat 423 cells. In contrast, nuclear Cdc25A overexpression caused dephosphorylation and nuclear retention of the proapoptotic transcription factor Forkhead in rhabdomyosarcoma-like 1 (FKHRL1) in human N.1 ovarian carcinoma cells. This resulted in the increased constitutive expression of the FKHRL1 targets Fas ligand and Bim, and promoted apoptosis. Thus, the Cdc25A oncogene, which was found to be frequently overexpressed in certain human cancers, can increase or decrease the susceptibility to apoptosis depending on the cell-type-specific subcellular distribution.
Asunto(s)
Apoptosis/fisiología , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismo , Fosfatasas cdc25/metabolismo , Animales , Apoptosis/efectos de los fármacos , Fusión Artificial Génica , Compartimento Celular , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead , Vectores Genéticos , Humanos , Proteínas del Tejido Nervioso , Ratas , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes/genética , Transformación Genética , Factor de Necrosis Tumoral alfa/farmacología , Fosfatasas cdc25/genéticaRESUMEN
A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5-10 microM range. Higher BR-concentrations (20 microM) predominantly induced necrosis which correlated with DNA strand breaks and subsequent depletion of ATP- and dATP levels. Replenishment of the ATP pool by addition of adenosine prevented necrosis and favoured apoptosis. This effect was not a pecularity of BR-treatment, but was reproduced with high concentrations of all trans-retinoic acid (120 microM) and cyanide (20 mM). Glucose was also capable to suppress necrosis and to favour apoptosis of HL-60 cells, which had been treated with necrotic doses of BR and cyanide. Apoptosis eliminates unwanted cells without affecting the microenvironment, whereas necrosis causes severe inflammation of surrounding tissues due to spillage of cell fluids into the peri-cellular space. Thus, the monitoring and maintenance of cellular energy pools during therapeutic drug treatment may help to minimize nonspecific side effects and to improve attempted drug effects.
Asunto(s)
Adenosina Trifosfato/fisiología , Antineoplásicos/toxicidad , Apoptosis , Necrosis , Nucleósidos/toxicidad , Adenosina/farmacología , Adenosina Trifosfato/análisis , Benzamidas/farmacología , Ensayo Cometa , Daño del ADN , Nucleótidos de Desoxiadenina/análisis , Nucleótidos de Desoxicitosina/análisis , Desoxirribonucleótidos/análisis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células HL-60 , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Cianuro de Potasio/antagonistas & inhibidores , Tretinoina/antagonistas & inhibidoresRESUMEN
The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis appeared dependent on Cdc25A expression and Cdc25A over-expression could substitute for Myc-triggered apoptosis. These findings suggested that an important downstream component of Myc-mediated apoptosis was identified. However and in contrast, we recently reported that during TNFalpha-induced apoptosis, which required c-Myc function, Cdc25A was down-regulated in a human carcinoma cell line. We now provide evidence that Cdc25A rendered the non-transformed rat embryonic cell line 423 refractory to apoptosis, which was induced by serum deprivation and in absence of detectable c-myc levels. The survival promoting activity of cdc25A was abolished upon infection of cells with a full-length cdc25A antisense construct. To identify the signaling proteins mediating the survival function of the phosphatase, cdc25A- and akt- over-expressing pooled clones were exposed to selected chemicals, which inhibit or activate key proteins in signaling pathways. Inhibition of apoptosis by SU4984, NF023 and Rapamycin placed Cdc25A and Akt function downstream of FGF.R, PDGF.R, and compensated G-protein- and PP2A- activity. Interestingly, upon treatment with LY-294002, cdc25A- and akt- over-expressing clones exhibited similar apoptotic patterns as control cells, which indicates that neither Akt- nor Cdc25A-mediated survival functions are dependent on PI.3 kinase activity in rat 423 cells. In cdc25A-overexpressing cells increased levels of serine 473 phosphorylated Akt were found, which co-precipitated with Cdc25A and Raf1. Since activation of proteins requires dephosphorylation of particular residues in addition to site-specific phosphorylation, the anti-apoptotic effect of Cdc25A might derive from its participation in a multimeric protein complex with phosphoAkt and Raf1, two prominent components of survival pathways.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Arabidopsis , Medio de Cultivo Libre de Suero/farmacología , Fosfatasas cdc25/fisiología , Animales , Línea Celular/efectos de los fármacos , Cromonas/farmacología , Citocinas/farmacología , Depresión Química , Doxiciclina/farmacología , Embrión de Mamíferos/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Genes myc , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Sustancias Macromoleculares , Morfolinas/farmacología , Péptidos/farmacología , Fosforilación , Piperazinas/farmacología , Proteínas de Plantas/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Canales de Potasio/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Proto-Oncogénicas c-raf/fisiología , Ratas , Proteínas Recombinantes de Fusión/fisiología , Sirolimus/farmacología , Suramina/análogos & derivados , Suramina/farmacología , TransfecciónAsunto(s)
Transformación Celular Neoplásica/genética , Proteínas Nucleares/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Animales , División Celular , Genes ras , Linfoma Anaplásico de Células Grandes/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Fragmentos de Péptidos , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
OBJECTIVE: Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (RR) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleotide synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitumor therapy. MATERIALS AND METHODS: HL-60 and K562 leukemia cells were treated with increasing doses of amidox and didox. Thereafter, the mode of cytotoxic drug action was determined by Hoechst 33258/propidium iodide (HO/PI) double staining, annexin binding, DNA fragmentation, and caspase activation. This was correlated to the decrease in dNTP levels. Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatidylserine were used to quantify apoptosis. RESULTS: Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 microM amidox or 250 microM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100% of the HL-60 cells died by apoptosis without an increase in necrosis. K562 cells were found to be resistant to amidox but not to didox. In HL-60 cells, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but not amidox-induced apoptosis, can be correlated with the decrease in dNTP levels. The results suggests that amidox induces several apoptosis mechanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox in K562 cells, and because amidox hardly induces apoptosis in this cell line, no caspase cleavage is observed. CONCLUSIONS: Didox triggers distinct apoptosis pathways in HL-60 and K562 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Caspasas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Oximas/farmacología , Ribonucleótido Reductasas/antagonistas & inhibidores , Anexina A5/metabolismo , Caspasa 8 , Caspasa 9 , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Gelsolina/metabolismo , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Células K562/efectos de los fármacos , Células K562/enzimología , Fosfatidilserinas/metabolismo , Proyectos Piloto , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
Two hypothetical concepts are discussed as means to cure cancer: 1) extinction of the neoplastic cell pool which forms the tumor and 2) induction of terminal differentiation to park tumor cells in growth arrest (1). Sodium butyrate (NaB) has been shown to promote differentiation of HL-60 cells to mature monocytes (2) or an eosinophil-commited HL-60 subline to eosinophils (3). NaB also triggers terminal differentiation of keratinocytes (4). However, application of NaB to colon carcinoma cells (but not to normal colon cells) induces apoptosis (5). Therefore, a pharmacological agent that elicits differentiation in one cell type can trigger an entirely different response, apoptosis, in another.
RESUMEN
Ribonucleotide reductase (RR) is the rate-limiting enzyme for the de novo synthesis of deoxyribonucleotides. Its activity is significantly increased in tumor cells related to the proliferation rate. Therefore, the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study, we investigated whether the antineoplastic effects of trimidox (3,4, 5-trihydroxybenzamidoxime), a novel inhibitor of RR, were due to induction of apoptosis.HL-60 cells were incubated with various concentrations of trimidox. Consequently, cell morphology, DNA condensation, annexin binding, DNA fragmentation, and signature type cleavage of poly(ADP-ribose)polymerase and gelsolin were determined. We also tested the involvement of CD95 and CD95 ligand in apoptosis induction. Furthermore, we examined the c-myc expression of HL-60 cells after incubation with trimidox in order to elucidate a possible association between c-myc expression and induction of apoptosis in the case of trimidox. Trimidox incubation caused a time-dependent increase of c-myc RNA expression and this was accompanied by the induction of apoptosis. Apoptosis was triggered independently of CD95 by the activation of caspases and PARP cleavage. We conclude that trimidox is able to induce programmed cell death. The induction of apoptosis was demonstrated by various biochemical and morphological methods and seems to be associated with the induction of c-myc. Apoptosis was induced by the activation of caspases and without change of the CD95 and CD95 ligand expression.