RESUMEN
Our understanding of cancer biology has increased substantially over the past 30 years. Despite this, and an increasing pharmaceutical company expenditure on research and development, the approval of novel oncology drugs during the past decade continues to be modest. In addition, the attrition of agents during clinical development remains high. This attrition can be attributed, at least in part, to the clinical development being underpinned by the demonstration of predictable efficacy in experimental models of human tumours. This review will focus on the range of models available for the discovery and development of anticancer drugs, from traditional subcutaneous injection of tumour cell lines to mice genetically engineered to spontaneously give rise to tumours. It will consider the best time to use the models, along with practical applications and shortcomings. Finally, and most importantly, it will describe how these models reflect the underlying cancer biology and how well they predict efficacy in the clinic. Developing a line of sight to the clinic early in a drug discovery project provides clear benefit, as it helps to guide the selection of appropriate preclinical models and facilitates the investigation of relevant biomarkers.
Asunto(s)
Antineoplásicos/uso terapéutico , Modelos Animales de Enfermedad , Desarrollo de Medicamentos , Descubrimiento de Drogas , Animales , Línea Celular Tumoral , Humanos , Ratones , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo. METHODS: The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling. RESULTS: RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53(wt) (U87MG, A172, and primary GBM2), and p53(mut) (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53(wt) and p53(mut) cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53(wt) and p53(mut) GBM cells. PRKD2 knockdown in p53(wt) cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53(mut) GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation. CONCLUSIONS: PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Senescencia Celular/fisiología , Glioma/metabolismo , Proteínas Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Silenciador del Gen , Xenoinjertos , Humanos , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Proteína Quinasa D2 , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , TransfecciónRESUMEN
Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-kappaB activation; and abrogated the expression of NF-kappaB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 micromol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-kappaB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pancreáticas/patología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Desnudos , Peso Molecular , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/tratamiento farmacológico , Estilbenos/farmacología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrosarcoma/metabolismo , Inmunohistoquímica , Ratones , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Isoformas de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Aptamers, also termed as decoys or "chemical antibodies," represent an emerging class of therapeutics. They are short DNA or RNA oligonucleotides or peptides that assume a specific and stable three-dimensional shape in vivo, thereby providing specific tight binding to protein targets. In some cases and as opposed to antisense oligonucleotides, effects can be mediated against extracellular targets, thereby preventing a need for intracellular transportation. The first aptamer approved for use in man is a RNA-based molecule (Macugen, pegaptanib) that is administered locally (intravitreally) to treat age-related macular degeneration by targeting vascular endothelial growth factor. The most advanced aptamer in the cancer setting is AS1411, formerly known as AGRO100, which is being administered systemically in clinical trials. AS1411 is a 26-mer unmodified guanosine-rich oligonucleotide, which induces growth inhibition in vitro, and has shown activity against human tumor xenografts in vivo. The mechanism underlying its antiproliferative effects in cancer cells seems to involve initial binding to cell surface nucleolin and internalization, leading to an inhibition of DNA replication. In contrast to other unmodified oligonucleotides, AS1411 is relatively stable in serum-containing medium, probably as a result of the formation of dimers and a quartet structure. In a dose escalation phase I study in patients with advanced solid tumors, doses up to 10 mg/kg/d (using a four or seven continuous infusion regime) have been studied. Promising signs of activity have been reported (multiple cases of stable disease and one near complete response in a patient with renal cancer) in the absence of any significant adverse effects. Further trials are ongoing in renal and non-small cell lung cancers. In preclinical studies, additional aptamers have been described against several cancer targets, such as tenascin-C, the transcription factor signal transducer and activator of transcription 3, and antiapoptotic and Ku proteins.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Aptámeros de Péptidos/farmacología , Neoplasias/tratamiento farmacológico , Animales , Aptámeros de Nucleótidos/uso terapéutico , Aptámeros de Péptidos/uso terapéutico , Humanos , Neoplasias/genética , Oligodesoxirribonucleótidos/farmacocinética , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/uso terapéuticoRESUMEN
The bis-sulfamoylated derivative of 2-methoxyestradiol (2-MeOE2), 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE), has shown potent antiproliferative and antiangiogenic activity in vitro and inhibits tumor growth in vivo. 2-MeOE2bisMATE is bioavailable, in contrast to 2-MeOE2 that has poor bioavailability. In this study, we have examined the role of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 in the metabolism of 2-MeOE2. In MDA-MB-231 cells, which express high levels of 17beta-HSD type 2, and in MCF-7 cells transfected with 17beta-HSD type 2, high-performance liquid chromatography analysis showed that a significant proportion of 2-MeOE2 was metabolized to inactive 2-methoxyestrone. Furthermore, MCF-7 cells transfected with 17beta-HSD type 2 were protected from the cytotoxic effects of 2-MeOE2. In contrast, no significant metabolism of 2-MeOE2bisMATE was detected in transfected cells and 17beta-HSD type 2 transfection did not offer protection against 2-MeOE2bisMATE cytotoxicity. This study may go some way to explaining the poor bioavailability of 2-MeOE2, as the gastrointestinal mucosa expresses high levels of 17beta-HSD type 2. In addition, this study shows the value of synthesizing sulfamoylated derivatives of 2-MeOE2 with C17-position modifications as these compounds have improved bioavailability and potency both in vitro and in vivo.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Neoplasias de la Mama/enzimología , Estradiol/análogos & derivados , 2-Metoxiestradiol , Biotransformación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Estradiol/metabolismo , Estradiol/farmacocinética , Humanos , Estereoisomerismo , TransfecciónRESUMEN
Steroid sulphatase inhibitors which decrease or prevent the biosynthesis of oestrogens, potentially have an important role in the treatment of breast cancer in postmenopausal women. The non-steroidal sulphatase inhibitor 667 COUMATE has been shown to be active both in vitro and in vivo. The pharmacokinetics of this drug have not been investigated. In preparation for the clinical evaluation of this agent, a sensitive and robust reversed phase high-performance liquid chromatography (HPLC) method was developed for the detection of 667 COUMATE in biological fluids. The sulphatase inhibitor was extracted from plasma with diethyl ether and separated from putative metabolites and endogenous plasma components with a C3-phenyl column. Using this method an extraction efficiency of 76+/-5% and a limit of detection of less than 0.1 ng/ml was achieved. The stability of this agent was investigated under different pH conditions and during storage in plasma at room temperature or -20 degrees C. 667 COUMATE was found to be stable when stored in acidified plasma (pH 4.5) at -20 degrees C. In conclusion, the HPLC method developed is a reproducible and sensitive assay that will enable quantitation of the potent non-steroidal sulphatase inhibitor 667 COUMATE in biological fluids in the forthcoming Phase I clinical trial.
Asunto(s)
Química Clínica/métodos , Cumarinas/análisis , Sulfonamidas/análisis , Cromatografía Líquida de Alta Presión , Éter/análisis , Humanos , Concentración de Iones de Hidrógeno , Modelos Químicos , Ácidos Sulfónicos , Temperatura , Factores de TiempoRESUMEN
Curcumin, the yellow pigment in turmeric, prevents malignancies in the intestinal tract of rodents. It is under clinical evaluation as a potential colon cancer chemopreventive agent. The systemic bioavailability of curcumin is low, perhaps attributable, at least in part, to metabolism. Indirect evidence suggests that curcumin is metabolized in the intestinal tract. To investigate this notion further, we explored curcumin metabolism in subcellular fractions of human and rat intestinal tissue, compared it with metabolism in the corresponding hepatic fractions, and studied curcumin metabolism in situ in intact rat intestinal sacs. Analysis by high-performance liquid chromatography, with detection at 420 or 280 nm, permitted characterization of curcumin conjugates and reduction products. Chromatographic inferences were corroborated by mass spectrometry. Curcumin glucuronide was identified in intestinal and hepatic microsomes, and curcumin sulfate, tetrahydrocurcumin, and hexahydrocurcumin were found as curcumin metabolites in intestinal and hepatic cytosol from humans and rats. The extent of curcumin conjugation was much greater in intestinal fractions from humans than in those from rats, whereas curcumin conjugation was less extensive in hepatic fractions from humans than in those from rats. The curcumin-reducing ability of cytosol from human intestinal and liver tissue exceeded that observed with the corresponding rat tissue by factors of 18 and 5, respectively. Curcumin sulfate was identified in incubations of curcumin with intact rat gut sacs. Curcumin was sulfated by human phenol sulfotransferase isoenzymes SULT1A1 and SULT1A3. Equine alcohol dehydrogenase catalyzed the reduction of curcumin to hexahydrocurcumin. The results show that curcumin undergoes extensive metabolic conjugation and reduction in the gastrointestinal tract and that there is more metabolism in human than in rat intestinal tissue. The pharmacological implications of the intestinal metabolism of curcumin should be taken into account in the design of future chemoprevention trials of this dietary constituent.