RESUMEN
Dimethylaminoparthenolide (DMAPT) is a water soluble parthenolide analog with preclinical activity in hematologic malignancies. Using non-small lung cancer (NSCLC) cell lines (A549 and H522) and an immortalized human bronchial epithelial cell line (BEAS2B) and TCC cell lines (UMUC-3, HT-1197 and HT-1376) and a bladder papilloma (RT-4), we aimed to characterize DMAPT's anticancer activity in tobacco-associated neoplasms. Flow cytometric, electrophoretic mobility gel shift assays (EMSA), and Western blot studies measured generation of reactive oxygen species (ROS), inhibition of NFκB DNA binding, and changes in cell cycle distribution and apoptotic proteins. DMAPT generated ROS with subsequent JNK activation and also decreased NFκB DNA binding and antiapoptotic proteins, TRAF-2 and XIAP. DMAPT-induced apoptotic cell death and altered cell cycle distribution with upregulation of p21 and p73 levels in a cell type-dependent manner. DMAPT suppressed cyclin D1 in BEAS2B. DMAPT retained NFκB and cell cycle inhibitory activity in the presence of the tobacco carcinogen nitrosamine ketone, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Using a BrdU accumulation assay, 5-20 µM of DMAPT was shown to inhibit cellular proliferation of all cell lines by more than 95%. Oral dosing of DMAPT suppressed in vivo A549 and UMUC-3 subcutaneous xenograft growth by 54% (p = 0.015) and 63% (p < 0.01), respectively, and A549 lung metastatic volume by 28% (p = 0.043). In total, this data demonstrates DMAPT's novel anticancer properties in both early and late stage tobacco-associated neoplasms as well as its significant in vivo activity. The data provides support for the conduct of clinical trials in TCC and NSCLC.
Asunto(s)
División Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , FN-kappa B/metabolismo , Nicotiana , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/farmacología , Neoplasias de la Vejiga Urinaria/patología , Animales , Secuencia de Bases , Carcinógenos/toxicidad , Ciclo Celular/efectos de los fármacos , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Desnudos , Nitrosaminas/toxicidad , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sesquiterpenos/química , Nicotiana/química , Neoplasias de la Vejiga Urinaria/etiología , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
BACKGROUND: To characterize the molecular changes associated with DMAPT-induced prostate cancer cell death and its in vivo activity. METHODS: CWR22Rv1 and PC-3 were subjected to flow cytometry, electrophoretic mobility shift assays, and Western blot studies to measure DMAPT's ability to generate reactive oxygen species (ROS), inhibit NFkappaB DNA binding, and cause changes in anti-apoptotic proteins. N-acetyl cysteine (NAC) and short hairpin RNA (shRNA) were used to determine the contribution of ROS and JNK2 activation, respectively. The BrdU incorporation assay was used to measure proliferation and trypan blue studies assessed cell viability after DMAPT treatment. The in vivo activity of DMAPT as a single agent and in combination with bicalutamide or docetaxel was assessed in a subcutaneous xenograft model with athymic nude female mice. RESULTS: DMAPT generated ROS with subsequent JNK activation and inhibited NFkappaB DNA binding and expression of NFkappaB-regulated anti-apoptotic proteins. DMAPT increased necrotic and apoptotic cell death in a cell-type-dependent manner and both types of cell death were blocked by NAC. Additionally, shRNA JNK2 partially blocked the anti-proliferative activity of DMAPT. DMAPT inhibited CWR22Rv1 and PC-3 cellular proliferation by 100% with 10 and 20 microM respectively and in vivo, DMAPT was more effective at inhibiting growth than biclutamide (CWR22v1) and docetaxel (PC-3). CONCLUSIONS: DMAPT promotes cell death by both generating ROS and inhibition of NFkappaB. Its in vivo activity supports the conduct of clinical trials in patients with castrate-resistant disease.
Asunto(s)
FN-kappa B/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/farmacología , Acetilcisteína/metabolismo , Animales , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Organismos Libres de Patógenos EspecíficosRESUMEN
Rapid assays for bacteria have been developed utilizing novel LysLysLys-isoluminol (14) and GluGlu-isoluminol (16) probes that have been derived from peptides which potentially mimic bacteriophage attachment protein binding regions. Compared to two conventional methods that are widely used, namely nucleic acid probes and polymerase chain reaction (PCR) assays, these types of probes may eventually have certain advantages, such as high sensitivity, and short preparation and assay time.
Asunto(s)
Bacterias/aislamiento & purificación , Dipéptidos/química , Colorantes Fluorescentes/química , Luminol/análogos & derivados , Oligopéptidos/química , Espectrometría de Fluorescencia/métodos , Proteínas Bacterianas/metabolismo , Dipéptidos/síntesis química , Colorantes Fluorescentes/síntesis química , Luminol/síntesis química , Luminol/química , Oligopéptidos/síntesis química , Unión Proteica , Factores de TiempoRESUMEN
A series of aminoparthenolide analogs (6-37) were synthesized and evaluated for their anti-leukemic activity. Eight compounds exhibited good anti-leukemic activity with LD(50)'s in the low microM range (1.5-3.0microM). Compounds 16, 24 and 30 were the most potent compounds in the series, causing greater than 90% cell death at 10microM concentration against primary AML cells in culture, with LD(50) values of 1.7, 1.8 and 1.6microM.
Asunto(s)
Antineoplásicos/síntesis química , Leucemia Mieloide Aguda/tratamiento farmacológico , FN-kappa B/metabolismo , Sesquiterpenos/síntesis química , Animales , Antineoplásicos/farmacología , Muerte Celular , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Lactonas/química , Modelos Químicos , Ratas , Sesquiterpenos/química , Sesquiterpenos/farmacología , Solubilidad , Agua/químicaRESUMEN
Crystals of the title salt, C(17)H(28)NO(3) (+)·C(4)H(3)O(4) (-), were obtained by reacting parthenolide with dimethyl-amine followed by conversion of the amine adduct into a water-soluble fumarate salt. Subsequent crystallization of the fumarate salt from water afforded colorless ortho-rhom-bic crystals. The amine addition is highly stereospecific yielding exclusively a single diastereomer with R-configuration at the newly formed C-11 chiral carbon. In the crystal, intermolecular O-Hâ¯O and N-Hâ¯O hydrogen bonds help to establish the packing.
RESUMEN
Nuclear factor-kappaB (NF-kappaB) has been implicated in a number of malignancies and has been suggested to be a potential molecular target in the treatment of leukaemia. This study demonstrated the constitutive activation of NF-kappaB in human myeloid blasts and a clear correlation between NF-kappaB expression and in vitro cytoprotection. High NF-kappaB expression was found in many of the poor prognostic acute myeloid leukaemia (AML) subtypes, such as French-American-British classification M0 and M7, and the poor cytogenetic risk group. The in vitro effects of LC-1, a novel dimethylamino-parthenolide analogue, were assessed in 62 primary untreated AML samples. LC-1 was found to be cytotoxic to AML cells in a dose-dependent manner, mediated through the induction of apoptosis. The median drug concentration necessary to kill 50% of the cells was 4.5 micromol/l for AML cells, compared with 12.8 micromol/l for normal marrow cells. LC-1 was shown to reduce the five individual human NF-kappaB Rel proteins in a dose-dependent manner. The subsequent inhibition of many NF-kappaB-regulated cytokines was also demonstrated. Importantly, sensitivity to LC-1 was correlated with the basal NF-kappaB activity. Consequently, LC-1 treatment provides a proof of principle for the use of NF-kappaB inhibitors in the treatment of AML.
Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , FN-kappa B/antagonistas & inhibidores , Sesquiterpenos/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , FN-kappa B/análisis , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Tumorales Cultivadas , Adulto JovenRESUMEN
OBJECTIVES: Cancer of the exocrine pancreas is the fourth leading cause of cancer-related deaths in the United States. The efficacy of a novel bioavailable anticancer agent, dimethylamino-parthenolide (DMAPT), and the cyclooxygenase 2 inhibitor, celecoxib, was evaluated in a carcinogen-induced developmental model of pancreatic cancer. METHODS: Syrian golden hamsters were injected with N-nitrosobis(2-oxopropyl)amine, once weekly for 6 weeks. Upon the first injection, hamsters were randomized as follows: placebo, low-/high-dose DMAPT (20 and 40 mg/kg per day), low-/high-dose celecoxib (10and 50 mg/kg per day), or combination DMAPT/celecoxib (low/low, high/high). RESULTS: The 32-week trial showed that 40 mg/kg DMAPT alone significantly decreased the size of gross pancreatic cancers relative to placebo. No significant difference in gross tumor number was observed between the treatment groups and placebo with the exception of 50 mg/kg celecoxib with a higher tumor incidence; this group also exhibited lower lymphotactin levels suggestive of decreased immune surveillance. Tumor invasion into adjacent organs and metastasis were not observed in the DMAPT/celecoxib treatment groups. Drug targets including prostaglandin E2, prostaglandin E2 metabolite and activated nuclear factor kappaB were significantly decreased. CONCLUSIONS: Dimethylamino-parthenolide and celecoxib have the potential to be novel chemotherapeutic agents for pancreatic cancer; however, further optimization or the use of other modalities may be required for chemoprevention.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Pancreáticas/prevención & control , Animales , Celecoxib , Proliferación Celular/efectos de los fármacos , Quimiocinas C/metabolismo , Cricetinae , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Antígeno Ki-67/metabolismo , Mesocricetus , Mucinas/metabolismo , FN-kappa B/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Nitrosaminas , Neoplasias Pancreáticas/inducido químicamente , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pirazoles/administración & dosificación , Sesquiterpenos/administración & dosificación , Sulfonamidas/administración & dosificación , Factores de TiempoRESUMEN
In this study, we characterized nuclear factor kappaB (NF-kappaB) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-kappaB. However, all cases showed higher basal NF-kappaB than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-kappaB induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P < .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD(50) of 2.8 microM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P < .001). Apoptosis was preceded by a marked loss of NF-kappaB DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r(2) = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r(2) = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/patología , Factor de Transcripción ReIA/metabolismo , Apoptosis , ADN/metabolismo , Progresión de la Enfermedad , Humanos , Unión Proteica , Factor de Transcripción ReIA/análisis , Células Tumorales CultivadasRESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer deaths in the United States. Inflammatory molecules, cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-kappaB) have been implicated in lung carcinogenesis. The therapeutic potential of celecoxib, a COX-2 selective inhibitor, and LC-1, a pro-apoptotic drug with accompanying inhibition of NF-kappaB, were investigated. MATERIALS AND METHODS: Syrian golden hamsters (n = 140) underwent N-nitroso-bis(2-oxopropyl)amine (BOP) injection weekly for 6 wk. Hamsters were randomized into seven groups: placebo and low/high doses of LC-1, celecoxib, and LC-1/celecoxib. Treatments were given via orogastric lavage for 32 wk. Immunohistochemistry was used to determine COX-2 expression and NF-kappaB activity. Ki-67 labeling was used as an index of proliferation. COX activity was measured by prostaglandin E(2) enzyme-linked immunosorbent assay. RESULTS: BOP successfully induced lung adenocarcinoma in 63% of placebo animals. Lung tumors strongly expressed COX-2 and NF-kappaB. Prostaglandin E(2) levels were decreased in celecoxib compared with placebo groups (P < 0.05) reflecting suppression of COX activity, but no decrease in NF-kappaB was seen as measured by immunohistochemistry in the tumors. There was no significant difference in tumor size, tumor incidence, or tumor proliferation index between placebo and treatment groups. CONCLUSIONS: Carcinogen exposure results in increased COX-2 and NF-kappaB expression and suggests a role in carcinogenesis. Celecoxib and LC-1 did not have any effect in preventing lung cancer development when co-administered with and continued after the carcinogen BOP. Higher doses that can result in suppression of NF-kappaB activity will need to be explored to determine the viability of this approach to prevent lung cancer development.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , Adenocarcinoma/inducido químicamente , Adenocarcinoma/metabolismo , Animales , Carcinógenos , Celecoxib , Cricetinae , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/metabolismo , Mesocricetus , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Nitrosaminas , Distribución Aleatoria , Sesquiterpenos/uso terapéuticoRESUMEN
Leukemia stem cells (LSCs) are thought to play a central role in the pathogenesis of acute leukemia and likely contribute to both disease initiation and relapse. Therefore, identification of agents that target LSCs is an important consideration for the development of new therapies. To this end, we have previously demonstrated that the naturally occurring compound parthenolide (PTL) can induce death of human LSCs in vitro while sparing normal hematopoietic cells. However, PTL has relatively poor pharmacologic properties that limit its potential clinical use. Consequently, we generated a family of PTL analogs designed to improve solubility and bioavailability. These studies identified an analog, dimethylamino-parthenolide (DMAPT), which induces rapid death of primary human LSCs from both myeloid and lymphoid leukemias, and is also highly cytotoxic to bulk leukemic cell populations. Molecular studies indicate the prevalent activities of DMAPT include induction of oxidative stress responses, inhibition of NF-kappaB, and activation of p53. The compound has approximately 70% oral bioavailability, and pharmacologic studies using both mouse xenograft models and spontaneous acute canine leukemias demonstrate in vivo bioactivity as determined by functional assays and multiple biomarkers. Therefore, based on the collective preclinical data, we propose that the novel compound DMAPT has the potential to target human LSCs in vivo.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Sesquiterpenos/farmacología , Animales , Disponibilidad Biológica , Perros , Diseño de Fármacos , Humanos , Ratones , FN-kappa B/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Sesquiterpenos/farmacocinética , Relación Estructura-Actividad , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
The design of novel targeted or combination therapies may improve treatment options for pancreatic cancer. Two targets of recent interest are nuclear factor-kappaB (NF-kappaB) and cyclooxygenase (COX), known to be activated or overexpressed, respectively, in pancreatic cancer. We have previously shown that parthenolide, a proapoptotic drug associated with NF-kappaB inhibition, enhanced the growth suppression of pancreatic cancer cells by the COX inhibitor sulindac in vitro. In the present study, a bioavailable analogue of parthenolide, LC-1, and sulindac were evaluated in vivo using a xenograft model of human pancreatic cancer. Treatment groups included placebo, low-dose/high-dose LC-1 (20 and 40 mg/kg), low-dose/high-dose sulindac (20 and 60 mg/kg), and low-dose combination LC-1/sulindac (20 mg/kg each). In MiaPaCa-2 xenografts, tumor growth was inhibited by either high-dose sulindac or LC-1. In BxPC-3 xenografts, tumor size was significantly reduced by treatment with the low-dose LC-1/sulindac combination or high-dose sulindac alone (P < 0.05). Immunohistochemistry of BxPC-3 tumors revealed a significant decrease in Ki-67 and CD31 staining by high-dose sulindac, with no significant changes in COX-1/COX-2 levels or activity in any of the treatment groups. NF-kappaB DNA-binding activity was significantly decreased by high-dose LC-1. Cyclin D1 protein levels were reduced by the low-dose LC-1/sulindac combination or high-dose sulindac alone, correlating with BxPC-3 tumor suppression. These results suggest that LC-1 and sulindac may mediate their antitumor effects, in part, by altering cyclin D1 levels. Furthermore, this study provides preclinical evidence for the therapeutic efficacy of these agents.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ciclina D1/antagonistas & inhibidores , Neoplasias Pancreáticas/patología , Sesquiterpenos/farmacología , Sulindac/farmacología , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , FN-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.