RESUMEN
Drug development, resource- and time-intensive, extensively employs cell-based assays to assess the efficacy and safety of candidate drugs. The widely used immortalized cell lines, experimentally convenient, have limited predictive value. In contrast, ex-vivo models more faithfully reproduce diseases but are technically challenging to establish. To address this need, we developed a simplified process for ex-vivo cell culture, demonstrating its feasibility in ocular surface cells. Conjunctival cells were harvested by impression cytology and grown on mixed cellulose ester membrane filters (MCFs). Human and rabbit conjunctival cells cultured on MCFs are 100% viable at 24 h, and 43% viable at 72 h. A gene expression study evaluating 84 genes involved in ocular inflammation demonstrated that ex-vivo culturing maintains intact the expression of two thirds of these genes in human cells. That these cells are suitable for the assessment of ocular drugs was demonstrated by studying the effect of phosphosulindac (PS), a small molecule under development for the treatment of dry eye disease, in both human and rabbit conjunctival cells. PS, for example, suppressed the expression of CXCL10, a cytokine participating in the pathogenesis of dry eye disease, in human and in rabbit conjunctival cells cultured ex-vivo by 32% and 70%, respectively. Conjunctival cells cultured ex-vivo can be transfected to evaluate mechanistic questions. We successfully transfected such cells with a plasmid expressing luciferase under the control of an IFN-γ-responsive promoter or its control plasmid. IFN-γ stimulated luciferase expression by 85% in cells with the responsive plasmid but not in controls; PS significantly suppressed this induction by 37% without affecting the control plasmid. These findings demonstrate that human and rabbit conjunctival cells cultured ex-vivo with our method are viable and maintain their biological integrity; respond to biological and pharmacological agents; and are transfectable with informative plasmids. The unique advantage of this method is to potentially accelerate the development of novel drugs for the treatment of ocular surface diseases, and to advance our understanding of ocular surface pathophysiology.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Conjuntiva/efectos de los fármacos , Evaluación de Medicamentos/métodos , Síndromes de Ojo Seco/tratamiento farmacológico , Compuestos Organofosforados/uso terapéutico , Sulindac/análogos & derivados , Adulto , Anciano , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Celulosa/análogos & derivados , Celulosa/química , Quimiocina CXCL10/metabolismo , Conjuntiva/metabolismo , Desarrollo de Medicamentos , Femenino , Perfilación de la Expresión Génica , Humanos , Luciferasas/metabolismo , Masculino , Persona de Mediana Edad , Plásmidos , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulindac/uso terapéutico , Obtención de Tejidos y Órganos , TransfecciónRESUMEN
New chemotherapeutic agents are needed for pancreatic cancer (PC). We have previously shown that phospho-valproic acid (MDC-1112) is effective in cell-line xenografts of PC. Here, we explored whether MDC-1112 is effective in additional clinically relevant animal models of PC and whether MDC-1112 enhances the anticancer effect of clinically used chemotherapeutic agents. MDC-1112 alone strongly reduced patient-derived pancreatic tumor xenograft growth, and extended survival of LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) mice. In both models, MDC-1112 inhibited STAT3 activation and its downstream signals, including Bcl-xL and cyclin D1. In human PC cell lines, P-V enhanced the growth inhibitory effect of gemcitabine (GEM), Abraxane and 5-FU, but not that of irinotecan. Normal human pancreatic epithelial cells were more resistant to the cytotoxic effects of MDC-1112/GEM combination. Furthermore, MDC-1112 enhanced GEM's effect on colony formation, apoptosis, cell migration, and cell invasion. In vivo, MDC-1112 and GEM, given alone, reduced patient-derived pancreatic tumor xenograft growth by 58% and 87%, respectively; whereas MDC-1112/GEM combination reduced tumor growth by 94%, inducing tumor stasis. In conclusion, MDC-1112 should be further explored as a potential agent to be used in combination with GEM for treating PC.
Asunto(s)
Anomalías Múltiples/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Queratocono/congénito , Organofosfatos/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Ácido Valproico/análogos & derivados , Anomalías Múltiples/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Humanos , Queratocono/tratamiento farmacológico , Queratocono/patología , Ratones , Neoplasias Pancreáticas/patología , Transducción de Señal/efectos de los fármacos , Ácido Valproico/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
New therapeutic strategies against glioblastoma multiforme (GBM) are urgently needed. Signal transducer and activator of transcription 3 (STAT3), constitutively active in many GBM tumors, plays a major role in GBM tumor growth and represents a potential therapeutic target. We have documented previously that phospho-valproic acid (MDC-1112), which inhibits STAT3 activation, possesses strong anticancer properties in multiple cancer types. In this study, we explored the anticancer efficacy of MDC-1112 in preclinical models of GBM, and evaluated its mode of action. MDC-1112 inhibited the growth of multiple human GBM cell lines in a concentration- and time-dependent manner. Normal human astrocytes were resistant to MDC-1112, indicating selectivity. In vivo, MDC-1112 reduced the growth of subcutaneous GBM xenografts in mice by up to 78.2% (P < 0.01), compared with the controls. Moreover, MDC-1112 extended survival in an intracranial xenograft model. Although all vehicle-treated mice died by 19 days of treatment, 7 of 11 MDC-1112-treated mice were alive and healthy by the end of 5 weeks, with many showing tumor regression. Mechanistically, MDC-1112 inhibited STAT3 phosphorylation at the serine 727 residue, but not at tyrosine 705, in vitro and in vivo. STAT3 overexpression rescued GBM cells from the cell growth inhibition by MDC-1112. In addition, MDC-1112 reduced STAT3 levels in the mitochondria and enhanced mitochondrial levels of reactive oxygen species, which triggered apoptosis. In conclusion, MDC-1112 displays strong efficacy in preclinical models of GBM, with the serine 727 residue of STAT3 being its key molecular target. MDC-1112 merits further evaluation as a drug candidate for GBM. New therapeutic options are needed for glioblastoma. The novel agent MDC-1112 is an effective anticancer agent in multiple animal models of glioblastoma, and its mechanism of action involves the inhibition of STAT3 phosphorylation, primarily at its Serine 727 residue.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Glioblastoma/patología , Organofosfatos/farmacología , Factor de Transcripción STAT3/metabolismo , Ácido Valproico/análogos & derivados , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Glioblastoma/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT3/efectos de los fármacos , Ácido Valproico/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Given that glioblastoma multiforme (GBM) is associated with poor prognosis, new agents are urgently needed. We developed phospho-glycerol-ibuprofen-amide (PGIA), a novel ibuprofen derivative, and evaluated its safety and efficacy in preclinical models of GBM, and its mechanism of action using human GBM cells and animal tumor models. Furthermore, we explored whether formulating PGIA in polymeric nanoparticles could enhance its levels in the brain. PGIA was 3.7- to 5.1-fold more potent than ibuprofen in suppressing the growth of human GBM cell lines. PGIA 0.75× IC50 inhibited cell proliferation by 91 and 87% in human LN-229 and U87-MG GBM cells, respectively, and induced strong G1/S arrest.In vivo, compared with control, PGIA reduced U118-MG and U87-MG xenograft growth by 77 and 56%, respectively (P< 0.05), and was >2-fold more efficacious than ibuprofen. Normal human astrocytes were resistant to PGIA, indicating selectivity. Mechanistically, PGIA reduced cyclin D1 levels in a time- and concentration-dependent manner in GBM cells and in xenografts. PGIA induced cyclin D1 degradation via the proteasome pathway and induced dephosphorylation of GSK3ß, which was required for cyclin D1 turnover. Furthermore, cyclin D1 overexpression rescued GBM cells from the cell growth inhibition by PGIA. Moreover, the formulation of PGIA in poly-(L)-lactic acid poly(ethylene glycol) polymeric nanoparticles improved its pharmacokinetics in mice, delivering PGIA to the brain. PGIA displays strong efficacy against GBM, crosses the blood-brain barrier when properly formulated, reaching the target tissue, and establishes cyclin D1 as an important molecular target. Thus, PGIA merits further evaluation as a potential therapeutic option for GBM.
Asunto(s)
Neoplasias Encefálicas/patología , Ciclina D1/metabolismo , Glioblastoma/patología , Ibuprofeno/análogos & derivados , Animales , Línea Celular Tumoral , Ibuprofeno/química , RatonesRESUMEN
Because cancer cell invasion is a critical determinant of metastasis, targeting invasion is a viable approach to prevent metastasis. Utilizing a novel three-dimensional high-throughput invasion assay, we screened a National Cancer Institute compound library and discovered compounds demonstrating inhibitory effects on cancer cell invasion. One hit, trifluoperazine, suppresses invasion of human cancer cell lines while displaying a limited cytotoxicity profile. This inhibition is due to the interference with cancer cell migratory ability but not proteolytic activity. Treatment of cancer cells with trifluoperazine significantly reduces angiogenesis and prevents cancer cell invasion through a chorioallantoic basement membrane. Mechanistically, treatment results in decreased phosphorylated AKT (Ser(473) and Thr(308)) and ß-catenin (Ser(552)). Lack of phosphorylation of Ser(552) of ß-catenin prevents ß-catenin nuclear relocation, resulting in decreased expression of vascular endothelial growth factor, likely mediated through dopamine receptor D2. Taken together, we demonstrated that trifluoperazine is responsible for reducing the angiogenic and invasive potential of aggressive cancer cells through dopamine receptor D2 to modulate the ß-catenin pathway and propose that trifluoperazine may be used as an antimetastasis chemotherapeutic.
Asunto(s)
Antineoplásicos/farmacología , Antipsicóticos/farmacología , Invasividad Neoplásica/prevención & control , Trifluoperazina/farmacología , Animales , Línea Celular Tumoral , Embrión de Pollo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Células 3T3 NIH , Invasividad Neoplásica/patologíaRESUMEN
Colorectal cancer (CRC) is a serious yet preventable disease. The low acceptance and cost of colonoscopy as a screening method or CRC make chemoprevention an important option. Nonsteroidal anti-inflammatory drugs (NSAIDs), not currently recommended for CRC prevention, have the potential to evolve into the agents of choice for this indication. Here, we discuss the promise and challenge of NSAIDs for this chemopreventive application.Multiple epidemiologic studies, randomized clinical trials (RCTs) of sporadic colorectal polyp recurrence, RCTs in patients with hereditary colorectal cancer syndromes, and pooled analyses of cardiovascular-prevention RCTs linked to cancer outcomes have firmly established the ability of conventional NSAIDs to prevent CRC. NSAIDs, however, are seriously limited by their toxicity,which can become cumulative with their long-term administration for chemoprevention, whereas drug interactions in vulnerable elderly patients compound their safety. Newer, chemically modified NSAIDs offer the hope of enhanced efficacy and safety.Recent work also indicates that targeting earlier stages of colorectal carcinogenesis, such as the lower complexity aberrant crypt foci, is a promising approach that may only require relatively short use of chemopreventive agents. Drug combination approaches exemplified by sulindac plus difluoromethylornithine appear very efficacious. Identification of those at risk or most likely to benefit from a given intervention using predictive biomarkers may usher in personalized chemoprevention. Agents that offer simultaneous chemoprevention of diseases in addition to CRC, e.g., cardiovascular and/or neurodegenerative diseases,may have a much greater potential for a broad clinical application.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Neoplasias Colorrectales/prevención & control , Antiinflamatorios no Esteroideos/efectos adversos , HumanosRESUMEN
PURPOSE: The purpose of the study was to evaluate the metabolism, pharmacokinetics and efficacy of phospho-NSAIDs in Ces1c-knockout mice. METHODS: Hydrolysis of phospho-NSAIDs by Ces1c was investigated using Ces1c-overexpressing cells. The rate of phospho-NSAID hydrolysis was compared between wild-type, Ces1c+/- and Ces1c-/- mouse plasma in vitro, and the effect of plasma Ces1c on the cytotoxicity of phospho-NSAIDs was evaluated. Pharmacokinetics of phospho-sulindac was examined in wild-type and Ces1c-/- mice. The impact of Ces1c on the efficacy of phospho-sulindac was investigated using lung and pancreatic cancer models in vivo. RESULTS: Phospho-NSAIDs were extensively hydrolyzed in Ces1c-overexpressing cells. Phospho-NSAID hydrolysis in wild-type mouse plasma was 6-530-fold higher than that in the plasma of Ces1c-/- mice. Ces1c-expressing wild-type mouse serum attenuated the in vitro cytotoxicity of phospho-NSAIDs towards cancer cells. Pharmacokinetic studies of phospho-sulindac using wild-type and Ces1c-/- mice demonstrated 2-fold less inactivation of phospho-sulindac in the latter. Phospho-sulindac was 2-fold more efficacious in inhibiting the growth of lung and pancreatic carcinoma in Ces1c -/- mice, as compared to wild-type mice. CONCLUSIONS: Our results indicate that intact phospho-NSAIDs are the pharmacologically active entities and phospho-NSAIDs are expected to be more efficacious in humans than in rodents due to their differential expression of carboxylesterases.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/uso terapéutico , Aspirina/análogos & derivados , Hidrolasas de Éster Carboxílico/genética , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Ibuprofeno/análogos & derivados , Organofosfatos/uso terapéutico , Compuestos Organofosforados/uso terapéutico , Sulindac/análogos & derivados , Animales , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacocinética , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Aspirina/metabolismo , Aspirina/farmacocinética , Aspirina/uso terapéutico , Hidrolasas de Éster Carboxílico/sangre , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Ibuprofeno/metabolismo , Ibuprofeno/farmacocinética , Ibuprofeno/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organofosfatos/metabolismo , Organofosfatos/farmacocinética , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacocinética , Sulindac/metabolismo , Sulindac/farmacocinética , Sulindac/uso terapéuticoRESUMEN
Esterase hydrolysis of drugs can accelerate their elimination, thereby limiting their efficacy. Polyethylene glycol (PEG) covalently attached to drugs (pegylation) is known to improve the efficiency of many drugs. Using as a test agent the novel phospho-ibuprofen (PI), we examined whether pegylation of PI could abrogate its hydrolytic degradation by esterases; PI, known to inhibit colon cancer growth, has a carboxylic ester hydrolyzable by carboxylesterases (CES). We covalently attached mPEG-2000 to PI (PI-PEG) and studied its stability by exposing it to cells overexpressing CES and by administering it to mice. We also evaluated PI-PEG's anticancer efficacy in human colon cancer xenografts and in Apc(min/+) mice. PI-PEG was stable in the presence of cells overexpressing CES1 or CES2, whereas PI was extensively hydrolyzed (90.2 ± 0.7%, 14.3 ± 1.1%, mean ± S.E.M.). In mice, PI was nearly completely hydrolyzed. Intravenous administration of PI-PEG resulted in significant levels in blood and in colon cancer xenografts (xenograft values in parentheses): area under the curve for 0-24 hours = 2351 (2621) (nmol/g) × h; Cmax = 1965 (886) nmol/g; Tmax = 0.08 (2) hour. The blood levels of ibuprofen, its main hydrolytic product, were minimal. Compared with controls, PI-PEG inhibited the growth of the xenografts by 74.8% (P < 0.01) and reduced intestinal tumor multiplicity in Apc(min/+) mice by 73.1% (P < 0.01), prolonging their survival (100% versus 55.1% of controls; P = 0.013). Pegylation protects PI from esterase hydrolysis and improves its pharmacokinetics. In preclinical models of colon cancer, PI-PEG is a safe and efficacious agent that merits further evaluation.
Asunto(s)
Antineoplásicos/farmacocinética , Ibuprofeno/análogos & derivados , Organofosfatos/farmacocinética , Polietilenglicoles/química , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Células HEK293 , Humanos , Hidrólisis , Ibuprofeno/efectos adversos , Ibuprofeno/síntesis química , Ibuprofeno/farmacocinética , Ibuprofeno/uso terapéutico , Ratones , Organofosfatos/efectos adversos , Organofosfatos/síntesis química , Organofosfatos/uso terapéutico , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we synthesized phospho-aspirin (PA-2; MDC-22), a novel derivative of aspirin, and evaluated its chemotherapeutic and chemopreventive efficacy in preclinical models of triple negative breast cancer (TNBC). METHODS: Efficacy of PA-2 was evaluated in human breast cancer cells in vitro, and in orthotopic and subcutaneous TNBC xenografts in nude mice. Mechanistic studies were also carried out to elucidate the mechanism of action of PA-2. RESULTS: PA-2 inhibited the growth of TNBC cells in vitro more potently than aspirin. Treatment of established subcutaneous TNBC xenografts (MDA-MB-231 and BT-20) with PA-2 induced a strong growth inhibitory effect, resulting in tumor stasis (79% and 90% inhibition, respectively). PA-2, but not aspirin, significantly prevented the development of orthotopic MDA-MB-231 xenografts (62% inhibition). Mechanistically, PA-2: 1) inhibited the activation of epidermal growth factor receptor (EGFR) and suppressed its downstream signaling cascades, including PI3K/AKT/mTOR and STAT3; 2) induced acetylation of p53 at multiple lysine residues and enhanced its DNA binding activity, leading to cell cycle arrest; and 3) induced oxidative stress by suppressing the thioredoxin system, consequently inhibiting the activation of the redox sensitive transcription factor NF-κB. These molecular alterations were observed in vitro and in vivo, demonstrating their relevance to the anticancer effect of PA-2. CONCLUSIONS: Our findings demonstrate that PA-2 possesses potent chemotherapeutic efficacy against TNBC, and is also effective in its chemoprevention, warranting further evaluation as an anticancer agent.
Asunto(s)
Aspirina/análogos & derivados , Receptores ErbB/antagonistas & inhibidores , Neoplasias Mamarias Experimentales/prevención & control , Organofosfatos/uso terapéutico , Estrés Oxidativo/fisiología , Proteína p53 Supresora de Tumor/administración & dosificación , Proteína p53 Supresora de Tumor/metabolismo , Acetilación/efectos de los fármacos , Animales , Aspirina/administración & dosificación , Aspirina/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/prevención & control , Línea Celular Tumoral , Receptores ErbB/fisiología , Femenino , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Organofosfatos/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
We have synthesized a novel derivative of indomethacin, phospho-tyrosol-indomethacin (PTI; MPI-621), and evaluated its anticancer efficacy in vitro and in vivo. PTI inhibited the growth of human colon, breast and lung cancer cell lines 6-30-fold more potently than indomethacin. In vivo, in contrast to indomethacin that was unable to inhibit colon cancer xenograft growth, PTI inhibited the growth of colon (69% at 10mg/kg/day, P < 0.01) and lung (91% at 15mg/kg/day, P < 0.01) subcutaneous cancer xenografts in immunodeficient mice, suppressing cell proliferation by 33% and inducing apoptosis by 75% (P < 0.05, for both). Regarding its pharmacokinetics in mice, after a single intraperitoneal injection of PTI, its plasma levels reached the maximum concentration (Cmax = 46 µM) at 2h (Tmax) and became undetectable at 4h. Indomethacin is the major metabolite of PTI, with plasma Cmax = 378 µM and Tmax = 2.5h; it became undetectable 24h postadministration. The cellular uptake of PTI (50-200 µM) at 6h was about 200-fold greater than that of indomethacin. Regarding its safety, PTI had no significant genotoxicity, showed less gastrointestinal toxicity than indomethacin and presented no cardiac toxicity. Mechanistically, PTI suppressed prostaglandin E2 production in A549 human lung cancer cells and strongly inhibited nuclear factor-κB activation in A549 xenografts. These findings indicate that PTI merits further evaluation as an anticancer agent.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Indometacina/análogos & derivados , Indometacina/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Organofosfatos/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dinoprostona/biosíntesis , Femenino , Humanos , Indometacina/sangre , Ratones , Ratones Desnudos , Ratones SCID , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , Trasplante de Neoplasias , Organofosfatos/sangre , Ratas , Ratas Sprague-Dawley , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We studied colon carcinogenesis using Fourier-transform infrared (FT-IR) microspectroscopy, an evolving method that allows the nondestructive assessment of the chemical composition of cells and tissues and of the in situ relationship between molecules, and assessed its diagnostic potential. Mid-FT-IR spectra were obtained from frozen colon tissue samples of normal (C57BL/6J) and Min (Apc(Min) mutant) mice, the latter recapitulating key features of human colon carcinogenesis. Classic spectroscopic analysis demonstrated marked differences in the Mid-FT-IR spectra between normal and dysplastic tissues, especially regarding peak positions and band intensity ratios in the regions 1800 to 985 cm(-1) and 3000 to 2700 cm(-1), reflecting changes in cellular nucleic acids, phosphates, and carbohydrates. Analysis of the spectra using the multivariate methods backpropagation neural networks, decision tree, adaboost with decision tree, and support vector machine, which interrogated the intrinsic dimensionality of IR spectra, revealed that their sensitivity was between 91.1% and 100% and their specificity between 94.1% and 100%, with the outcomes of the Support Vector Machine algorithm being identical to those of histologic analysis. FT-IR microspectroscopy holds great promise not only as a method of ascertaining changes in the chemistry of the neoplastic cells but also as a diagnostic tool, especially for early stages of carcinogenesis not detectable by other means.
Asunto(s)
Transformación Celular Neoplásica/química , Transformación Celular Neoplásica/patología , Colon/patología , Neoplasias del Colon/diagnóstico , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Neoplasias del Colon/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Análisis Multivariante , Máquina de Vectores de SoporteRESUMEN
Pharmacological targeting of breast cancer stem cells (CSCs) is highly promising for the treatment of breast cancer, as the small population of CSCs appears responsible for tumor initiation and progression and also for resistance to conventional treatment. Here we report that the novel phosphosulindac (OXT-328, PS) selectively and effectively eliminates breast CSCs both in vitro and in vivo. PS reduced cell proliferation and induced apoptosis in various breast CSCs. Breast CSCs are resistant to conventional cancer drugs but are sensitive to PS. Long-term treatment of mixtures of cultured breast CSCs and breast cancer cells with PS preferentially eliminated the CSCs. PS impaired the ability of CSCs to form mammospheres and markedly suppressed the expression of CSC-related genes. More importantly, PS prevented by half (p = .06) the formation of tumors initiated by CSCs in immunodeficient mice, and inhibited by 83% (p < .05) the growth of already formed breast cancer xenografts, reducing the proportion of CSCs in them. PS suppressed the Wnt/ß-catenin pathway by stimulating the degradation of ß-catenin and its relocalization to the cell membrane and also blocked the epithelial-mesenchymal transition and the generation of breast CSCs. These results indicate that PS has a strong inhibitory effect against breast cancer, acting, at least in part, by targeting CSCs through a signaling mechanism involving Wnt signaling.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Huésped Inmunocomprometido , Células Madre Neoplásicas/efectos de los fármacos , Compuestos Organofosforados/farmacología , Sulindac/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Técnicas de Cocultivo , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Terapia Molecular Dirigida , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Sulindac/farmacología , Proteínas Wnt/genética , Proteínas Wnt/inmunología , Vía de Señalización Wnt/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/inmunologíaRESUMEN
PURPOSE: Formulate phospho-sulindac (P-S, OXT-328) in a Pluronic hydrogel to be used as a topical anti-inflammatory agent and study its efficacy, safety and pharmacokinetics in an arthritis model. METHODS: LEW/crlBR rats with Freund's adjuvant-induced arthritis were treated with P-S formulated in Pluronic hydrogel (PSH). We determined the clinical manifestations of arthritis including the locomotor activity of the rats; evaluated joints for inflammation, bone resorption, cartilage damage, COX-2 expression and NF-κB activation; assayed plasma IL-6 and IL-10 levels; and studied the pharmacokinetics of P-S in rats after topical or oral administration. RESULTS: PSH applied at the onset of arthritis or when arthritis was fully developed, suppressed it by 56-82%, improved the locomotor activity of the rats 2.1-4.4 fold, suppressed synovial inflammation, bone resorption, cartilage damage, NF-κB activation and COX-2 expression but not plasma IL-6 and IL-10 levels. There were no side effects. PSH produced rapidly high local levels of P-S with <14% of P-S reaching the circulation, while orally administered P-S was rapidly metabolized generating much lower joint levels of P-S. CONCLUSIONS: Topical application of PSH is efficacious and safe in the treatment of Freund's adjuvant-induced arthritis; has a favorable pharmacokinetic profile; and likely acts by suppressing key pro-inflammatory signaling pathways.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Hidrogel de Polietilenoglicol-Dimetacrilato/administración & dosificación , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacocinética , Compuestos Organofosforados/administración & dosificación , Compuestos Organofosforados/farmacocinética , Sulindac/análogos & derivados , Sulindac/administración & dosificación , Sulindac/farmacocinética , Administración Oral , Administración Tópica , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacocinética , Artritis Experimental/sangre , Artritis Experimental/inducido químicamente , Artritis Experimental/metabolismo , Resorción Ósea/sangre , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Interleucina-10/sangre , Interleucina-10/metabolismo , Interleucina-6/sangre , Interleucina-6/metabolismo , Articulaciones/efectos de los fármacos , Articulaciones/metabolismo , Actividad Motora/efectos de los fármacos , FN-kappa B/metabolismo , Ratas , Ratas Endogámicas Lew , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismoRESUMEN
Purpose: To evaluate the hypothesis that 3 novel compounds, OXT-328, Q-922, and CL-717 show efficacy in the treatment of oxygen-induced retinopathy (OIR) and whether or not their route of administration is intravitreal, topical, or systemic. Methods: The OIR mouse model, characterized by an avascular area (AVA) and a neovascular area (NVA) of the retina, was used to study retinopathy of prematurity and other retinal diseases characterized by abnormal vessel growth. We measured the effect of our compounds on both the AVA and NVA in whole mounts of mouse retinal tissue. We also evaluated their ability to prevent new vessel formation in chicken chorioallantoic membranes (CAMs). Finally, we measured the in vitro uptake and biodistribution of topically applied CL-717 in human eye explants. Results: In mice with OIR, compared to controls, a single intravitreal administration of Q-922 or OXT-328 significantly reduced both AVA and NVA. CL-717 administered as eye drops over 5 days also reduced AVA and NVA, whereas OXT-328 eye drops had no effect. Q-922 given intraperitoneal (150 mg/kg/day × 5 days) reduced AVA and NVA. Remarkably, explanted human eyes bathed in CL-717 show rapid uptake and biodistribution in ocular tissues. In the chicken CAM model, all 3 compounds reduced the formation of new blood vessels by about one-third. No side effect in mice was observed, except for mild ocular surface irritation with Q-922. Conclusions: Systemic administration of Q-922 or topical administration of CL-717 holds particular promise for a simplified treatment of proliferative retinopathies without the necessity of intravitreal injections.
Asunto(s)
Enfermedades de la Retina , Neovascularización Retiniana , Retinopatía de la Prematuridad , Humanos , Animales , Ratones , Recién Nacido , Oxígeno , Vasos Retinianos , Animales Recién Nacidos , Distribución Tisular , Enfermedades de la Retina/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/tratamiento farmacológico , Retinopatía de la Prematuridad/tratamiento farmacológicoRESUMEN
Background: Chemotherapy-induced peripheral neuropathy (CIPN), a side effect of chemotherapy, is particularly difficult to treat. We explored whether phosphosulindac (PS), a modified NSAID, could treat CIPN. Methods: CIPN was induced in male C57BL/6 J mice by paclitaxel, vincristine or oxaliplatin. Mechanical allodynia was measured with the von Frey test and cold allodynia with the acetone test. To determine the preventive effect of PS, it was administered 2 days before the induction of CIPN. Mouse Lewis lung carcinoma xenografts were used to determine if PS altered the chemotherapeutic efficacy of paclitaxel. Cultured cell lines were used to evaluate the effect of PS on neuroinflammation. Results: Treatment with each of the three chemotherapeutic agents used to induce CIPN lowered the mechanical allodynia scores by 56 to 85% depending on the specific agent. PS gel was applied topically 3x/day for 16-22 days to the hind paws of mice with CIPN. This effect was dose-dependent. Unlike vehicle, PS returned mechanical allodynia scores back to pre-CIPN levels. PS had a similar effect on paclitaxel-induced CIPN cold allodynia. Sulindac, a metabolite of PS, had no effect on CIPN. PS significantly prevented CIPN compared to vehicle. Given concomitantly with paclitaxel to mice with lung cancer xenografts, PS relieved CIPN without affecting the anticancer effect of paclitaxel. The enantiomers of PS were equally efficacious against CIPN, suggesting the therapeutic suitability of the racemate PS. There were no apparent side effects of PS. PS suppressed the levels of IL-6, IL-10, CXCL1, and CXCL2 induced by paclitaxel in a neuroblastoma cell line, and macrophage activation to the M1 proinflammatory phenotype. Conclusion: Topically applied PS demonstrated broad therapeutic and preventive efficacy against CIPN, preserved the anticancer effect of paclitaxel, and was safe. Its anti-CIPN effect appears to be mediated, in part, by suppression of neuroinflammation. These data support further evaluation of topical PS for the control of CIPN.
RESUMEN
INTRODUCTION: We have recently synthesized phospho-ibuprofen (P-I; MDC-917), a safer derivative of ibuprofen, which has shown anti-cancer activity. We investigated its efficacy and mechanism of action in the treatment of breast cancer in preclinical models. METHODS: We evaluated the anti-breast-cancer efficacy of P-I alone or incorporated into liposomes (Lipo-P-I) in human estrogen receptor-positive (MCF-7) and triple-negative, i.e., estrogen receptor-negative, progesterone receptor-negative and HER2-negative (MDA-MB231) breast cancer cell lines - as they represent the most frequent (estrogen receptor-positive) and the most difficult-to-treat (triple-negative) subtypes of breast cancer - and their xenografts in nude mice. We assessed the effect of P-I on the levels of reactive oxygen nitrogen species in response to P-I using molecular probes, on the thioredoxin system (expression and redox status of thioredoxin-1 (Trx-1) and thioredoxin reductase activity), on cyclooxygenase 2, NF-κB and mitogen-activated protein kinase cell signaling; and on the growth of xenografts with stably knocked-down Trx-1. RESULTS: Compared with controls, P-I 400 mg/kg/day inhibited the growth of MDA-MB231 xenografts by 266%, while the growth of MCF-7 xenografts was inhibited 51% byP-I 300 mg/kg/day and 181% by Lipo-P-I 300 mg/kg/day. In both cell lines, P-I induced oxidative stress and suppressed the thioredoxin system (oxidized Trx-1 and decreased its expression; inhibited thioredoxin reductase activity). These changes triggered downstream redox signaling: the activity of NF-κB was suppressed and the Trx-1-ASK1 complex was dissociated, activating the p38 and JNK mitogen-activated protein kinase cascades. Trx-1 knockdown abrogated the anti-cancer effect of P-I in vitro and in vivo. CONCLUSION: P-I is safe and effective against breast cancer. Liposomal formulation enhances its efficacy; the effect is heavily dependent on the induction of oxidative stress and the suppression of the thioredoxin system. P-I merits further evaluation as an agent for the treatment of breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Ibuprofeno/análogos & derivados , Organofosfatos/farmacología , Tiorredoxinas/metabolismo , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Femenino , Glutatión/metabolismo , Humanos , Ibuprofeno/administración & dosificación , Ibuprofeno/farmacología , Liposomas , MAP Quinasa Quinasa Quinasa 5/metabolismo , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Organofosfatos/administración & dosificación , Oxidación-Reducción , Unión Proteica , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the current paradigm of anticancer drug development, candidate compounds are evaluated by testing their in vitro potency against molecular targets relevant to carcinogenesis, their effect on cultured cancer cells, and their ability to inhibit cancer growth in animal models. We discuss the key assumptions inherent in these approaches. In recent years, great emphasis has been placed on selecting for development compounds with nanomolar in vitro potency, expecting that they will be efficacious and safer based on the assumption that they can be used at lower doses ("the nanomolar rule"). However, this rule ignores critical parameters affecting efficacy and toxicity such as physiochemical and absorption, distribution, metabolism and excretion properties, off-target effects, and multitargeting activities. Thus, uncritical application of the nanomolar rule may reject efficacious compounds or select ineffective or toxic compounds. We present examples of efficacious chemotherapeutic (alkylating agents, hormonal agents, antimetabolites, thalidomide, and valproic acid) and chemopreventive (aspirin and sulindac) agents having millimolar potency and compounds with nanomolar potency (cyclooxygenase-2 inhibitors) that, nevertheless, failed or proved to be unsafe. The effect of candidate drugs on animal models of cancer is a better predictor of human drug efficacy; particularly useful are tumor xenografts. Given the cost of failure at clinical stages, it is imperative to keep in mind the limitations of the nanomolar rule and use relevant in vivo models early in drug discovery to prioritize candidates. Although in vivo models will continue having a major role in cancer drug development, more robust approaches that combine high predictive ability with simplicity and low cost should be developed.
Asunto(s)
Antineoplásicos/uso terapéutico , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Nanopartículas/administración & dosificación , Neoplasias/tratamiento farmacológico , Animales , Humanos , Modelos Animales , Modelos Moleculares , Tamaño de la PartículaRESUMEN
Phospho-nonsteroidal anti-inflammatory drugs (phospho-NSAIDs) are novel NSAID derivatives with improved anticancer activity and reduced side effects in preclinical models. Here, we studied the metabolism of phospho-NSAIDs by carboxylesterases and assessed the impact of carboxylesterases on the anticancer activity of phospho-NSAIDs in vitro and in vivo. The expression of human liver carboxylesterase (CES1) and intestinal carboxylesterase (CES2) in human embryonic kidney 293 cells resulted in the rapid intracellular hydrolysis of phospho-NSAIDs. Kinetic analysis revealed that CES1 is more active in the hydrolysis of phospho-sulindac, phospho-ibuprofen, phospho-naproxen, phospho-indomethacin, and phospho-tyrosol-indomethacin that possessed a bulky acyl moiety, whereas the phospho-aspirins are preferentially hydrolyzed by CES2. Carboxylesterase expression leads to a significant attenuation of the in vitro cytotoxicity of phospho-NSAIDs, suggesting that the integrity of the drug is critical for anticancer activity. Benzil and bis-p-nitrophenyl phosphate (BNPP), two carboxylesterase inhibitors, abrogated the effect of carboxylesterases and resensitized carboxylesterase-expressing cells to the potent cytotoxic effects of phospho-NSAIDs. In mice, coadministration of phospho-sulindac and BNPP partially protected the former from esterase-mediated hydrolysis, and this combination more effectively inhibited the growth of AGS human gastric xenografts in nude mice (57%) compared with phospho-sulindac alone (28%) (p = 0.037). Our results show that carboxylesterase mediates that metabolic inactivation of phospho-NSAIDs, and the inhibition of carboxylesterases improves the efficacy of phospho-NSAIDs in vitro and in vivo.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Carboxilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Organofosfatos/metabolismo , Organofosfatos/farmacología , Animales , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/uso terapéutico , Aspirina/análogos & derivados , Aspirina/metabolismo , Aspirina/farmacología , Carboxilesterasa/antagonistas & inhibidores , Carboxilesterasa/genética , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Hidrolasas de Éster Carboxílico/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas/fisiología , Quimioterapia Combinada/métodos , Inhibidores Enzimáticos/farmacología , Femenino , Células HEK293 , Humanos , Hidrólisis , Ibuprofeno/análogos & derivados , Ibuprofeno/metabolismo , Ibuprofeno/farmacología , Indometacina/análogos & derivados , Indometacina/metabolismo , Indometacina/farmacología , Concentración 50 Inhibidora , Cinética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Naproxeno/análogos & derivados , Naproxeno/metabolismo , Naproxeno/farmacología , Nitrofenoles/farmacología , Nitrofenoles/uso terapéutico , Organofosfatos/sangre , Organofosfatos/uso terapéutico , Compuestos Organofosforados/sangre , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacología , Compuestos Organofosforados/uso terapéutico , Fenilglioxal/análogos & derivados , Fenilglioxal/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Sulindac/análogos & derivados , Sulindac/sangre , Sulindac/metabolismo , Sulindac/farmacología , Sulindac/uso terapéuticoRESUMEN
PURPOSE: To incorporate phospho-ibuprofen (P-I), a lipophilic, water insoluble novel anti-cancer agent, into pegylated liposomes and upon formulation optimization to evaluate its antitumor activity in vitro and in vivo. METHODS: P-I loaded liposomes were prepared using the thin-film hydration method, and characterized for size, zeta potential, drug content and drug release. We examined their physical stability by particle size changes; their lyophilization ability in the presence of cryoprotectants; and their antitumor activity in vitro in human cancer cell lines and in vivo in a xenograft murine model. RESULTS: P-I was successfully loaded into liposomes consisting of soy-PC and PEG(2000)-PE. These liposomes were <150 nm in diameter; exhibited prolonged stability in suspension and can be lyophilized using sucrose as cryoprotectant. P-I liposomes inhibited the growth of human cancer cell lines in vitro and in vivo of xenograft in nude mice to a greater extent than free P-I. CONCLUSIONS: High levels of P-I can be incorporated into liposomes which can be lyophilized in the presence of sucrose and showed good stability upon storage. Moreover, these drug-incorporating liposomes were capable of inhibiting the growth of xenografted tumors in mice more effectively than free P-I. These results justify further development of the P-I liposomes.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Ibuprofeno/análogos & derivados , Lípidos/química , Organofosfatos/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Neoplasias del Colon/patología , Crioprotectores/química , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Liofilización , Humanos , Ibuprofeno/administración & dosificación , Ibuprofeno/química , Ibuprofeno/farmacología , Concentración 50 Inhibidora , Inyecciones Intraperitoneales , Cinética , Liposomas , Ratones , Ratones SCID , Organofosfatos/administración & dosificación , Organofosfatos/química , Tamaño de la Partícula , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Solubilidad , Propiedades de Superficie , Tecnología Farmacéutica/métodos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To investigate the metabolism of phospho-aspirin (PA, MDC-22), a novel anti-cancer and anti-inflammatory agent. METHODS: The metabolism of PA was studied in the liver and intestinal microsomes from mouse, rat and human. RESULTS: PA is rapidly deacetylated to phospho-salicylic acid (PSA), which undergoes regioselective oxidation to generate 3-OH-PSA and 5-OH-PSA. PSA also can be hydrolyzed to give salicylic acid (SA), which can be further glucuronidated. PA is far more stable in human liver or intestinal microsomes compared to those from mouse or rat due to its slowest deacetylation in human microsomes. Of the five major human cytochrome P450 (CYP) isoforms, CYP2C19 and 2D6 are the most active towards PSA. In contrast to PSA, conventional SA is not appreciably oxidized by the CYPs and liver microsomes, indicating that PSA is a preferred substrate of CYPs. Similarly, PA, in contrast to PSA, cannot be directly oxidized by CYPs and liver microsomes, indicating that the acetyl group of PA abrogates its oxidation by CYPs. CONCLUSIONS: Our findings establish the metabolism of PA, reveal significant inter-species differences in its metabolic transformations, and provide an insight into the role of CYPs in these processes.