RESUMEN
In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.
Asunto(s)
Fibrosarcoma/patología , Receptores de Hialuranos/metabolismo , Interleucina-33/metabolismo , Neoplasias Pulmonares/patología , Macrófagos/inmunología , Glicoproteínas de Membrana/metabolismo , Células Madre Neoplásicas/patología , Animales , Apoptosis , Proliferación Celular , Fibrosarcoma/etiología , Fibrosarcoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/genética , Interleucina-33/genética , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos DBA , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Sarcoma Experimental/etiología , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Spinal cord injury (SCI) is an acute neurodegenerative disorder caused by traumatic damage of the spinal cord. The neuropathological evolution of the primary trauma involves multifactorial processes that exacerbate the pathology, worsening the neurodegeneration and limiting neuroregeneration. This complexity suggests that multi-therapeutic approaches, rather than any single treatment, might be more effective. Encouraging preclinical results indicate that stem cell-based treatments may improve the disease outcome due to their multi-therapeutic ability. Mesenchymal Stem Cells (MSCs) are currently considered one of the most promising approaches. Significant improvement in the behavioral outcome after MSC treatment sustained by hydrogel has been demonstrated. However, it is still not known how hydrogel contribute to the delivery of factors secreted from MSCs and what factors are released in situ. Among different mediators secreted by MSCs after seeding into hydrogel, we have found CCL2 chemokine, which could account for the neuroprotective mechanisms of these cells. CCL2 secreted from human MSCs is delivered efficaciously in the lesioned spinal cord acting not only on recruitment of macrophages, but driving also their conversion to an M2 neuroprotective phenotype. Surprisingly, human CCL2 delivered also plays a key role in preventing motor neuron degeneration in vitro and after spinal cord trauma in vivo, with a significant improvement of the motor performance of the rodent SCI models.
Asunto(s)
Biomimética , Quimiocina CCL2/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Traumatismos de la Médula Espinal/terapia , Animales , Quimiocina CCL2/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Hidrogeles , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/patología , Recuperación de la Función , Resultado del TratamientoRESUMEN
This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Trabectedina/farmacología , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Biología de Sistemas/métodos , Transcripción Genética/efectos de los fármacosRESUMEN
Trabectedin and its analogue lurbinectedin are effective drugs used in the treatment of ovarian cancer. Since the presence of ascites is a frequent event in advanced ovarian cancer we asked the question whether ascites could modify the activity of these compounds against ovarian cancer cells. The cytotoxicity induced by trabectedin or lurbinectedin against A2780, OVCAR-5 cell lines or primary culture of human ovarian cancer cells was compared by performing treatment in regular medium or in ascites taken from either nude mice or ovarian cancer patients. Ascites completely abolished the activity of lurbinectedin at up to 10nM (in regular medium corresponds to the IC90), strongly reduced that of trabectedin, inhibited the cellular uptake of lurbinectedin and, to a lesser extent, that of trabectedin. Since α1-acid glycoprotein (AGP) is present in ascites at relatively high concentrations, we tested if the binding of the drugs to this protein could be responsible for the reduction of their activity. Adding AGP to the medium at concentration range of those found in ascites, we reproduced the anticytotoxic effect of ascites. Erythromycin partially restored the activity of the drugs, presumably by displacing them from AGP. Equilibrium dialysis experiments showed that both drugs bind AGP, but the affinity of binding of lurbinectedin was much greater than that of trabectedin. KD values are 8±1.7 and 87±14nM for lurbinectedin and trabectedin, respectively. The studies intimate the possibility that AGP present in ascites might reduce the activity of lurbinectedin and to a lesser extent of trabectedin against ovarian cancer cells present in ascites. AGP plasma levels could influence the distribution of these drugs and thus they should be monitored in patients receiving these compounds.
Asunto(s)
Ascitis/metabolismo , Carbolinas/metabolismo , Dioxoles/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Orosomucoide/metabolismo , Neoplasias Ováricas/metabolismo , Tetrahidroisoquinolinas/metabolismo , Animales , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Carbolinas/farmacología , Carbolinas/uso terapéutico , Línea Celular Tumoral , Dioxoles/farmacología , Dioxoles/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/cirugía , Unión Proteica/fisiología , Tetrahidroisoquinolinas/farmacología , Tetrahidroisoquinolinas/uso terapéutico , Trabectedina , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Alzheimer's disease is a neurodegenerative disorder linked to oligomerization and fibrillization of amyloid ß peptides, with Aß1-42 being the most aggregative and neurotoxic one. We report herein the synthesis and conformational analysis of Aß1-42-amyloid related ß-hairpin peptidomimetics, built on a piperidine-pyrrolidine semi rigid ß-turn inducer and bearing two small recognition peptide sequences, designed on oligomeric and fibril structures of Aß1-42. According to these peptide sequences, a stable ß-hairpin or a dynamic equilibrium between two possible architectures was observed. These original constructs are able to greatly delay the kinetics of Aß1-42 aggregation process as demonstrated by thioflavin-T fluorescence, and transmission electron microscopy. Capillary electrophoresis indicates their ability to preserve the monomer species, inhibiting the formation of toxic oligomers. Furthermore, compounds protect against toxic effects of Aß on neuroblastoma cells even at substoichiometric concentrations. This study is the first example of acyclic small ß-hairpin mimics possessing such a highly efficient anti-aggregation activity. The protective effect is more pronounced than that observed with molecules which have undergone clinical trials. The structural elements made in this study provide valuable insights in the understanding of the aggregation process and insights to explore the design of novel acyclic ß-hairpin targeting other types of amyloid-forming proteins.
RESUMEN
BACKGROUND: In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. METHODS: Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. RESULTS: 402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. CONCLUSIONS: Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.
Asunto(s)
Antineoplásicos/farmacología , Dioxoles/farmacología , Compuestos Organoplatinos/farmacología , Tetrahidroisoquinolinas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Resistencia a Antineoplásicos , Femenino , Histonas/metabolismo , Humanos , Ratones , Ratones Desnudos , Trabectedina , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Staphylococcus aureus possesses two canonical ABC-importers dedicated to nickel acquisition: the NikABCDE and the CntABCDF systems, active under different growth conditions. This study reports on the extracytoplasmic nickel-binding components SaNikA and SaCntA. We showed by protein crystallography that SaNikA is able to bind either a Ni-(l-His)2 complex or a Ni-(l-His) (2-methyl-thiazolidine dicarboxylate) complex, depending on their availability in culture supernatants. Native mass spectrometry experiments on SaCntA revealed that it binds the Ni(ii) ion via a different histidine-dependent chelator but it cannot bind Ni-(l-His)2. In vitro experiments are consistent with in vivo nickel content measurements that showed that l-histidine has a high positive impact on nickel import via the Cnt system. These results suggest that although both systems may require free histidine, they use different strategies to import nickel.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Histidina/química , Níquel/química , Staphylococcus aureus/metabolismo , Tiazolidinas/química , Proteínas Bacterianas/química , Quelantes/química , Cristalografía , Citoplasma/metabolismo , Escherichia coli/metabolismo , Espectrometría de Masas , Conformación ProteicaRESUMEN
Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Dioxoles/farmacología , Liposarcoma Mixoide/genética , Liposarcoma Mixoide/metabolismo , Tetrahidroisoquinolinas/farmacología , Apoptosis , Proteína C-Reactiva/genética , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Metilación de ADN , Metilasas de Modificación del ADN/deficiencia , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/deficiencia , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos , Fibronectinas/genética , Humanos , Liposarcoma Mixoide/tratamiento farmacológico , Liposarcoma Mixoide/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Componente Amiloide P Sérico/genética , Transducción de Señal , Temozolomida , Trabectedina , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Rayos UltravioletaRESUMEN
The p73 locus gene has a complex structure encoding a plethora of isoforms. The different DeltaN truncated isoforms of p73 may exert different activities depending on the cellular context. The beta isoform of DeltaNp73 seems to have a particular pattern of action even if its role in cell cycle and mitosis is still under investigation. To gain further knowledge of DeltaNp73beta's function, we investigated the effects of its over-expression in tumour cellular models, using the tetracycline-inducible expression system. In the human lung carcinoma cell line H1299, DeltaNp73beta over-expression resulted in suppression of cell growth and in cell death. Surprisingly stable over-expression of DeltaNp73beta impaired the genomic stability of tumour cells, leading to the formation of tetraploid cells. The cells become enlarged and multinucleate, with incorrect mitotic figures, and died by apoptotic-independent pathways. Our data suggest that DeltaNp73beta-induced aberrant mitosis evades the control of the mitotic spindle assay checkpoint, leading to tetraploidy and cell death through mitotic catastrophe rather than apoptosis. The various C-terminal regions of DeltaNp73 may influence the final cellular phenotype and we assume that the beta one in particular could be important in both cell growth control and regulation of mitosis.
Asunto(s)
Apoptosis/genética , Proteínas de Unión al ADN/genética , Mitosis/genética , Proteínas Nucleares/genética , Poliploidía , Carcinoma Pulmonar de Células Pequeñas/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/fisiología , Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Trabectedin (ET-743, Yondelis) is a natural marine compound with antitumour activity currently undergoing phase II/III clinical trials. The mechanism of the drug's action is still to be defined, even though it has been clearly demonstrated the key role of Nucleotide Excision Repair (NER). To get further insights into the drug's mode of action, we studied the involvement of the DNA-double strand break repair (DNA-DSB) pathways: homologous and non-homologous recombination, both in budding yeasts and in mammalian cells and the possible cross-talk between NER and these repair pathways. Budding yeasts and mammalian cells deficient in the non-homologous end-joining pathway were moderately sensitive to trabectedin, while systems deficient in the homologous recombination pathway were extremely sensitive to the drug, with a 100-fold decrease in the IC50, suggesting that trabectedin-induced lesions are repaired by this pathway. The induction of Rad51 foci and the appearance of gamma-H2AX were chosen as putative markers for DNA-DSBs and were studied at different time points after trabectedin treatment in NER proficient and deficient systems. Both were clearly detected only in the presence of an active NER, suggesting that the DSBs are not directly caused by the drug, but are formed during the processing/repair of the drug- induced lesions.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Dioxoles/farmacología , Tetrahidroisoquinolinas/farmacología , Animales , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Células CHO , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cisplatino/farmacología , Cricetinae , Cricetulus , Reparación del ADN/genética , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Histonas/efectos de los fármacos , Histonas/metabolismo , Humanos , Inmunohistoquímica , Metilmetanosulfonato/farmacología , Mutación , Fosforilación , Saccharomyces cerevisiae/genética , TrabectedinaRESUMEN
T acute lymphoblastic leukemia cell lines treated with hexamethylene bisacetamide (HMBA) undergo a delay in cell cycle progression and increase susceptibility to apoptosis, although they never overcome the differentiation block. In accordance with changes in cell cycle and apoptosis, transitory p53 pathway activation commonly occurs. Bcl-2 inhibition further favours the pro-apoptotic effect of HMBA. Notch1 expression is down regulated by reduction of its transcription level. Accordingly, Notch1 protein and transcriptional activity were affected. Even if HMBA generally reduces Notch1 level in T acute lymphoblastic leukemia (T-ALL) cell lines, this does not commonly influence the biological response; in fact all the analysed cell lines, except CEM cells, display no biological effect following DAPT-induced Notch inhibition.
Asunto(s)
Acetamidas/farmacología , Antineoplásicos/farmacología , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/análisis , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptor Notch1/fisiología , Transducción de Señal , Triglicéridos/farmacología , Proteína p53 Supresora de Tumor/fisiología , Ácido gamma-Aminobutírico/análogos & derivados , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
OBJECTIVES: Trabectedin (ET-743, Yondelis) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)-rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug-induced cell cycle perturbations. MATERIALS AND METHODS: An isogenic NER-proficient cellular system (CHO-AA8) and a NER-deficient one (CHO-UV-96), lacking functional ERCC-1, were studied. Flow cytometric assays showed progressive accumulation of cells in G2 + M phase in NER-proficient but not in NER-deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. RESULTS: Cells exposed to trabectedin during G1 and G2 + M first experienced a G1 block, while those exposed in S phase were delayed in S and G2 + M phases but eventually divided. In the presence of functional NER, exit from the G1 block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G2 + M phase. This G2 + M processing of trabectedin-induced damage in NER-proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. CONCLUSIONS: This might be due either to important damage left unrepaired by previous G1 repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV-96 cells repair mechanisms other than NER are activated both in G1 and G2 + M phases.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Reparación del ADN , Dioxoles/farmacología , Modelos Biológicos , Tetrahidroisoquinolinas/farmacología , Animales , Bromodesoxiuridina/metabolismo , Células CHO , Recuento de Células , Muerte Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Simulación por Computador , Cricetinae , Cricetulus , ADN/biosíntesis , Citometría de Flujo , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Fase S/efectos de los fármacos , TrabectedinaRESUMEN
Integrin signalling co-ordinates with signalling originating from growth factor receptors in the co-operative control of cell proliferation, survival and migration. Increasing evidence suggests that integrins form physical complexes at the cell membrane with growth factor receptors, giving rise to signalling platforms at the adhesive sites. It is probable that at these sites integrins regulate adhesion and at the same time physically constrain and direct the response to soluble growth factors towards proliferation or survival stimuli. These co-operative effects might depend on integrin ability to activate growth factor receptors. In the present paper, we summarize our recent study showing that integrin-dependent adhesion triggers ligand-independent EGFR (epidermal growth factor receptor) activation to transduce downstream signalling. In addition, we also show that integrin-induced signalling pathways are necessary for EGF-dependent transcriptional response, demonstrating the requirement of the co-operation between cell-matrix adhesion and EGFR to achieve full biological responses.
Asunto(s)
Receptores ErbB/metabolismo , Integrinas/metabolismo , Animales , Adhesión Celular , Línea Celular , Factor de Crecimiento Epidérmico/metabolismo , Matriz Extracelular/metabolismo , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Mutación , Unión Proteica , Transducción de Señal , Transcripción GenéticaRESUMEN
It was previously suggested that the peculiar mechanism of action of the novel anticancer drug Yondelis (ET-743, trabectedin) was due to part of the molecule, units A and B, binding to DNA in the minor groove, causing an alkylation at the N2 of guanine, while unit C protrudes out of DNA, possibly interacting with transcription factors or other DNA binding proteins. To test this hypothesis, we have compared the biological activity and the mode of action of Yondelis with its analogue ET-637, which has the same chemical structure except for the lack of the C ring. Yondelis and ET-637 showed similar cytotoxic potency and cell cycle perturbations. As already reported for Yondelis, the UV-96 cell line, deficient in ERCC-1, was less sensitive to ET-637 than the parental cell line. The binding of Yondelis or ET-637 to DNA-oligonucleotides was demonstrated by gel shift assay and SDS did not reverse the binding. Both compounds blocked the temperature-induced activation of the HSP40 promoter in the range of 1-10 nM. This study indicates that ET-637 acts similarly to Yondelis and demonstrates that the C ring of Yondelis may not be required for its biological activity.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Dioxoles/farmacología , Isoquinolinas/farmacología , Antineoplásicos Alquilantes/química , Ciclo Celular/efectos de los fármacos , ADN/metabolismo , Reparación del ADN , Dioxoles/química , Femenino , Humanos , Isoquinolinas/química , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , Temperatura , Tetrahidroisoquinolinas , Trabectedina , Células Tumorales CultivadasRESUMEN
This paper illustrates some biochemical and pharmacological properties of two natural marine products such as trabectedin (ET-743, Yondelis) and aplidine. Trabectedin has shown clinical antitumor activity in refractory soft tissue sarcoma and ovarian cancer. The lack of cross resistance of trabectedin with other chemotherapeutic drugs is presumably related to its peculiar mode of action, that seems to be related to a promoter-dependent transcription modulation. Aplidine is a potent pro-apoptotic inducer in human leukemia and has antiangiogenic properties. These examples support the view that more research should be carried out to investigate new natural marine products since there are compounds among them with antitumor properties. Some of them appear to act by mechanisms different from those of conventional chemotherapeutic drugs and thus may be effective against tumors for which no active drugs are available.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Depsipéptidos/farmacología , Dioxoles/farmacología , Isoquinolinas/farmacología , Preparaciones Farmacéuticas/química , Animales , Antineoplásicos Alquilantes/química , Depsipéptidos/química , Dioxoles/química , Humanos , Isoquinolinas/química , Biología Marina , Péptidos Cíclicos , Sensibilidad y Especificidad , Relación Estructura-Actividad , Tetrahidroisoquinolinas , Trabectedina , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Yondelis (trabectidin, ET-743) is a marine natural product that has shown activity both in preclinical systems and in human malignancies such as soft tissue sarcoma and ovarian cancers that are resistant to previous chemotherapies. Molecular pharmacological studies indicated that Yondelis interacts with DNA and DNA repair systems in a way that is different from Cisplatin (DDP). The current study was designed to investigate the effects of the combination of Yondelis and DDP in human cancer cell lines and in xenografts derived from different tumours. The in vitro studies performed in human TE-671 rhabdomyosarcoma, Igrov-1 and 1A9 human ovarian carcinoma cell lines showed additive effects or slight synergism. Several human tumour xenografts, such as TE-671 rhabdomyosarcoma, SK-N-DX neuroblastoma, FADU head and neck, LX-1 non-small cell lung cancer (NSCLC), H-187 melanoma and SKOV HOC 8 ovarian carcinoma, showed an antitumour effect for the combination that was greater than that of each drug when given as a single agent. No consistent changes in the activity were observed if Yondelis and DDP were given 1 h apart in sequence or simultaneously. An orthotopically transplanted human ovarian cancer HOC 8 growing in the peritoneal cavity of nude mice was used that is insensitive to Yondelis alone and only moderately sensitive to DDP alone. The combination of the two drugs produced a dramatic increase of survival lasting several months. In conclusion, the combination of Yondelis and DDP is synergistic in vivo (i.e. the antitumour effect is greater than that of each drug used as a single agent at the maximum tolerated dose (MTD)) in different human tumour xenografts. The two drugs can be combined at the MTD of each drug, thus indicating there are no overlapping toxicities. These results provide a rationale for testing the combination of Yondelis and DDP in the clinic.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Rabdomiosarcoma/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Dioxoles/administración & dosificación , Dioxoles/efectos adversos , Sinergismo Farmacológico , Femenino , Humanos , Isoquinolinas/administración & dosificación , Isoquinolinas/efectos adversos , Ratones , Trasplante de Neoplasias , Tetrahidroisoquinolinas , Trabectedina , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The cytotoxic effect of Aplidin was investigated on fresh leukaemia cells derived from children with B-cell-precursor (BCP) acute lymphoblastic leukaemia (ALL) by using stromal-layer culture system and on four cell lines, ALL-PO, Reh, ALL/MIK and TOM-1, derived from patients with ALL with different molecular genetic abnormalities. In ALL cell lines Aplidin was cytotoxic at nanomolar concentrations. In the ALL cell lines the drug-induced cell death was clearly related to the induction of apoptosis and appeared to be p53-independent. Only in ALL-PO 20 nM Aplidin treatment caused a block of vascular endothelial growth factor (VEGF) secretion and downregulation of VEGF-mRNA, but Aplidin cytotoxicity does not seem to be related to VEGF inhibition since the sensitivity of ALL-PO cells to Aplidin is comparable to that observed for the other cells used. Aplidin induced a G(1) and a G(2) M block in ALL cell lines. In patient-derived leukaemia cells, Aplidin induced a strong cytotoxicity evidenced in a stroma-supported immunocytometric assay. Cells from children with genetic abnormalities such as t(9;22) and t(4;11) translocations, associated with an inferior treatment outcome, were sensitive to Aplidin to the same extent as that observed in other BCP-ALL cases. Aplidin exerted a strong cell killing effect (>88%) against primary culture cells from five relapsed ALL cases, at concentrations much lower than those reported to be achieved in plasma of patients receiving Aplidin at recommended doses. Taken together these data suggest that Aplidin could be a new anticancer drug to be investigated in ALL patients resistant to available therapy.
Asunto(s)
Antineoplásicos/farmacología , Depsipéptidos , Resistencia a Antineoplásicos , Péptidos Cíclicos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cariotipificación , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Espectrometría de Masas , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The mechanism by which aplidine, a marine natural product in early clinical development as an anticancer agent, induces cell growth inhibition and apoptosis has been investigated in the human leukemia cell line MOLT-4. This cell line is characterized not only by the ability to secrete VEGF, but also for the presence on its surface of the VEGF receptor-1 (VEGFR-1). Previous studies from our laboratory concerned with evaluating early changes in gene expression induced by aplidine in MOLT-4 cells have shown that the drug decreases the expression of VEGFR-1 (Marchini et al. Proc Am Assoc Cancer Res 2000; 41: 833). Here, we report the ability of aplidine to block the VEGF/VEGFR-1 loop. We found that aplidine blocked VEGF secretion that was temporally followed by a decrease in both VEGF and VEGFR-1 production. Aplidine did not directly affect either VEGF transcription or stabilization of its mRNA. Transfection of MOLT-4 cells with an antisense VEGF cDNA construct, resulted in inhibition of colony formations. One clone, transfected with sense VEGF cDNA, secreting 8-10 times more VEGF than parental cells, was less sensitive to aplidine-induced cytotoxicity and apoptosis than control cells. Moreover, addition of VEGF in the medium decreased the activity of aplidine in MOLT-4 cells. These data demonstrate that aplidine inhibits the growth and induces apoptosis in MOLT-4 cells through the inhibition of VEGF secretion which blocks the VEGF/VEGFR-1 autocrine loop necessary for the growth of these cells.
Asunto(s)
Antineoplásicos/farmacología , Depsipéptidos , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Leucemia de Células T/tratamiento farmacológico , Linfocinas/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Comunicación Autocrina , División Celular/efectos de los fármacos , Cartilla de ADN/química , Dactinomicina/farmacología , Ensayo de Cambio de Movilidad Electroforética , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Semivida , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Luciferasas/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Transducción de Señal , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial VascularRESUMEN
The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent "energy transfer" from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 micro g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.
Asunto(s)
Apoptosis , Supervivencia Celular , Citometría de Flujo/métodos , Leucemia de Células T/patología , Microscopía Fluorescente/métodos , Línea Celular Tumoral , ADN de Neoplasias/análisis , Citometría de Flujo/instrumentación , Humanos , Microscopía Fluorescente/instrumentación , PloidiasRESUMEN
Aplidine, dehydrodidemnin B, is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans currently in phase II clinical trial. In human Molt-4 leukaemia cells Aplidine was found to be cytotoxic at nanomolar concentrations and to induce both a G(1) arrest and a G(2) blockade. The drug-induced cell cycle perturbations and subsequent cell death do not appear to be related to macromolecular synthesis (protein, RNA, DNA) since the effects occur at concentrations (e.g. 10 nM) in which macromolecule synthesis was not markedly affected. Ten nM Aplidine for 1 h inhibited ornithine decarboxylase activity, with a subsequently strong decrease in putrescine levels. This finding has questionable relevance since addition of putrescine did not significantly reduce the cell cycle perturbations or the cytotoxicity of Aplidine. The cell cycle perturbations caused by Aplidine were also not due to an effect on the cyclin-dependent kinases. Although the mechanism of action of Aplidine is still unclear, the cell cycle phase perturbations and the rapid induction of apoptosis in Molt-4 cells appear to be due to a mechanism different from that of known anticancer drugs.