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1.
Transl Oncol ; 9(3): 228-35, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27267841

RESUMO

Induction of oxidative stress is a key component of cancer therapy. Pro-oxidant drugs have been demonstrated to enhance the efficacy of radiotherapy and chemotherapy. An emerging concept is that therapeutic outcomes are dictated by the differential redox buffering reserve in subpopulations of malignant cells, indicating the need for noninvasive biomarkers of tumor redox that can be used for dose identification and response assessment in a longitudinal setting. Magnetic resonance imaging (MRI) enhanced with the thiol-binding contrast agent Gd-LC6-SH, and hemodynamic response imaging (HRI) in combination with hypercapnia and hyperoxia were investigated as biomarkers of the pharmacodynamics of the small molecule pro-oxidant imexon (IMX). Human multiple myeloma cell lines 8226/S and an IMX-resistant variant, 8226/IM10, were established as contralateral tumors in SCID mice. T1slope, an MRI measure of the washout rate of Gd-LC6-SH, was significantly lower post-IMX therapy in 8226/S tumors compared with vehicle controls, indicating treatment-related oxidization of the tumor microenvironment, which was confirmed by analysis of tumor tissue for thiols. T1slope and ex vivo assays for thiols both indicated a more reduced microenvironment in 8226/IM10 tumors following IMX therapy. HRI with hypercapnia challenge revealed IMX inhibition of vascular dilation in 8226/S tumors but not 8226/IM10 tumors, consistent with decreased immunohistochemical staining for smooth muscle actin in treated 8226/S tumors. MRI enhanced with Gd-LC6-SH, and HRI coupled with a hypercapnic challenge provide noninvasive biomarkers of tumor response to the redox modulator imexon.

2.
Am J Transl Res ; 7(2): 348-55, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25901201

RESUMO

Bone is a favored site for solid tumor metastasis, especially among patients with breast, lung or prostate carcinomas. Micro CT is a powerful and inexpensive tool that can be used to investigate tumor progression in xenograft models of human disease. Many previous studies have relied on terminal analysis of harvested bones to document metastatic tumor activity. The current protocol uses live animals and combines sequential micro CT evaluation of lesion development with matched histopathology at the end of the study. The approach allows for both rapid detection and evaluation of bone lesion progression in live animals. Bone resident tumors are established either by direct (intraosseous) or arterial (intracardiac) injection, and lesion development is evaluated for up to eight weeks. This protocol provides a clinically relevant method for investigating bone metastasis progression and the development of osteotropic therapeutic strategies for the treatment of bone metastases.

3.
Cancer Biol Ther ; 16(1): 43-51, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25485960

RESUMO

Gemcitabine based treatment is currently a standard first line treatment for patients with advanced pancreatic cancer, however overall survival remains poor, and few options are available for patients that fail gemcitabine based therapy. To identify potential molecular targets in gemcitabine refractory pancreatic cancer, we developed a series of gemcitabine resistant (GR) cell lines. Initial drug exposure selected for an early resistant phenotype that was independent of drug metabolic pathways. Prolonged drug selection pressure after 16 weeks, led to an induction of cytidine deaminase (CDA) and enhanced drug detoxification. Cross resistance profiles demonstrate approximately 100-fold cross resistance to the pyrimidine nucleoside cytarabine, but no resistance to the same in class agents, azacytidine and decitabine. GR cell lines demonstrated a dose dependent collateral hypersensitivity to class I and II histone deacetylase (HDAC) inhibitors and decreased expression of 3 different global heterochromatin marks, as detected by H4K20me3, H3K9me3 and H3K27me3. Cell morphology of the drug resistant cell lines demonstrated a fibroblastic type appearance with loss of cell-cell junctions and an altered microarray expression pattern, using Gene Ontology (GO) annotation, consistent with progression to an invasive phenotype. Of particular note, the gemcitabine resistant cell lines displayed up to a 15 fold increase in invasive potential that directly correlates with the level of gemcitabine resistance. These findings suggest a mechanistic relationship between chemoresistance and metastatic potential in pancreatic carcinoma and provide evidence for molecular pathways that may be exploited to develop therapeutic strategies for refractory pancreatic cancer.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Inibidores de Histona Desacetilases/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Cromatina/genética , Cromatina/metabolismo , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fenótipo
4.
Blood ; 124(8): 1259-65, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-25016003

RESUMO

Lymphoma cells are subject to higher levels of oxidative stress compared with their normal counterparts and may be vulnerable to manipulations of the cellular redox balance. We therefore designed a phase 2 study of imexon (Amplimexon/NSC-714597), a prooxidant molecule, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Imexon was administered at 1000 mg/m(2) IV daily for 5 days in 21-day cycles. Gene expression analysis performed on pretreatment tumor specimens included 13 transcripts used to generate a redox signature score, previously demonstrated to correlate with lymphoma prognosis. Twenty-two patients were enrolled having follicular (n = 9), diffuse large B-cell (DLBCL) (n = 5), mantle cell (n = 3), transformed follicular (n = 2), small lymphocytic (n = 2), and Burkitt (n = 1) lymphoma. The most common grade 3/4 adverse events were anemia (14%) and neutropenia (9%). The overall response rate was 30%, including responses in follicular lymphoma (4 of 9) and DLBCL (2 of 5). Gene expression analyses revealed CD68 and the redox-related genes, GPX1 and SOD2, as well as a higher redox score to correlate with clinical responses. Therefore, pretreatment markers of oxidative stress may identify patients likely to respond to this therapeutic approach. This trial was registered at www.clinicaltrials.gov as #NCT01314014.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hexanonas/administração & dosagem , Oxidantes/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Intervalo Livre de Doença , Feminino , Glutationa Peroxidase/biossíntese , Hexanonas/efeitos adversos , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Oxidantes/efeitos adversos , Recidiva , Superóxido Dismutase/biossíntese , Taxa de Sobrevida
5.
Mol Cancer Ther ; 13(6): 1558-66, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24739392

RESUMO

Laminin-binding integrin receptors are key mediators of epithelial cell migration and tumor metastasis. Recent studies have demonstrated a role for the α6 integrin (ITGA6/CD49f) in maintaining stem cell compartments within normal bone marrow and in residency of tumors metastatic to bone. In this study, we tested a function-blocking antibody specific for ITGA6, called J8H, to determine if preexisting cancer lesions in bone could be slowed and/or animal survival improved. Human prostate tumors were established by intracardiac injection into male SCID mice and treatment with J8H antibody was initiated after 1 week. Tumor progression was monitored by micro-computed tomography (CT) imaging of skeletal lesions. Animals that received weekly injections of the anti-ITGA6 antibody showed radiographic progression in only 40% of osseous tumors (femur or tibia), compared with control animals, where 80% of the lesions (femur or tibia) showed progression at 5 weeks. Kaplan-Meier survival analysis demonstrated a significant survival advantage for J8H-treated animals. Unexpectedly, CT image analysis revealed an increased proportion of bone lesions displaying a sclerotic rim of new bone formation, encapsulating the arrested lytic lesions in animals that received the anti-ITGA6 antibody treatment. Histopathology of the sclerotic lesions demonstrated well-circumscribed tumor within bone, surrounded by fibrosis. These data suggest that systemic targeting of the ITGA6-dependent function of established tumors in bone may offer a noncytotoxic approach to arrest the osteolytic progression of metastatic prostate cancer, thereby providing a new therapeutic strategy for advanced disease.


Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos/administração & dosagem , Neoplasias Ósseas/tratamento farmacológico , Integrina alfa6/metabolismo , Terapia de Alvo Molecular , Neoplasias da Próstata/tratamento farmacológico , Animais , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Humanos , Integrina alfa6/efeitos dos fármacos , Masculino , Camundongos , Osteoblastos/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Invest New Drugs ; 31(6): 1616-25, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24037082

RESUMO

Microtubule targeting agents are among the most widely used chemotherapeutics for both solid and hematological malignancies. This study characterizes the diaryl-oxazole based anticancer agent PC-046, which was originally identified for development based on selective activity in deleted in pancreas cancer locus 4 (DPC4/SMAD4) deficient tumors. PC-046 has growth inhibitory activity in a variety of tumor types in vitro, and efficacy in SCID mice was shown in human tumor xenografts of MV-4-11 acute myeloid leukemia, MM.1S multiple myeloma, and DU-145 prostate cancer. Pharmacokinetic studies demonstrated relatively high oral bioavailability (71%) with distribution to both plasma and bone marrow. No myelosuppression was seen in non-tumor bearing SCID mice given a single dose just under the acute lethal dose. The COMPARE algorithm in the NCI-60 cell line panel demonstrated that PC-046 closely correlated to other known tubulin destabilizing agents (correlation coefficients ≈0.7 for vincristine and vinblastine). Mechanism of action studies showed cell cycle arrest in metaphase and inhibition of tubulin polymerization. Overall, these studies show that PC-046 is a synthetically-derived, small molecule microtubule destabilizing agent. Advantages over existing microtubule destabilizing agents include ease of synthesis, lack of MDR cross-resistance, good oral bioavailability and the lack of acute myelotoxicity.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Oxazóis/uso terapêutico , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Hematológicas/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Oxazóis/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Piridinas/farmacologia , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Cancer Chemother Pharmacol ; 71(6): 1567-76, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23572175

RESUMO

PURPOSE: Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo. METHODS: Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects. RESULTS: Inhibition of the Ca(2+)-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Calpaína/antagonistas & inibidores , Inibidores da Protease de HIV/farmacologia , Nelfinavir/farmacologia , Pirazinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Ácidos Borônicos/uso terapêutico , Bortezomib , Calpaína/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores da Protease de HIV/uso terapêutico , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/patologia , Nelfinavir/uso terapêutico , Pirazinas/uso terapêutico , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Invest New Drugs ; 31(3): 576-86, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23179338

RESUMO

Deletions or mutations in the tumor suppressor gene DPC4 (deleted in pancreatic carcinoma locus 4) are common in colon and pancreatic cancers. Using the Target-related Affinity Profiling (TRAP) chemical library screening method, a novel agent, UA8967, was selected for further studies because it showed greater potency in DPC4-deleted HCT-116 colon cancer cells. Cytotoxicity studies in six pancreatic cancer cell lines (MiaPaca-2, Panc-1, BxPC3, CF-PAC1, AsPC1, and T3M4), one normal human pancreatic ductal epithelial line (HPDE-6) and the HCT-116 DPC4(+/+) and HCT-116 DPC4(-/-) colon cancer cells showed IC50s ranging from 12-61 µM for exposure times of 72 h. Analysis of schedule dependence showed no advantage for long drug exposure times. There was also no selective inhibition of DNA, RNA or protein synthesis after exposure to UA8967. At 24-48 h, there was an accumulation of cells in G0/G1-phase and a proportionate reduction in S-phase cells. Within 1-6 h of exposure, cells were found to undergo an autophagic response, followed at 24 h by a low level of caspase-independent apoptosis with some necrosis. Because of the relatively non-specific mechanistic effects of UA8967, plasma membrane viability was evaluated using uptake of trypan blue and Sytox® Green dyes, and leakage of LDH. There was a dose dependent increase in Sytox® Green staining, trypan blue uptake and LDH leakage with increasing concentrations of UA8967, suggesting that UA8967 is affecting the plasma membrane. The DPC4(-/-) cells were more sensitive to UA8967 but not to DMSO, suggesting a drug-specific effect on cell membrane integrity.


Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Piperazinas/farmacologia , Proteína Smad4/genética , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos
9.
Mol Cancer Res ; 10(3): 392-400, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22275514

RESUMO

Oxidative protein folding in the endoplasmic reticulum (ER) requires strict regulation of redox homeostasis. Disruption of the lumenal redox balance induces an integrated ER stress response that is associated with reduced protein translation, increased chaperone activity, and ultimately cell death. Imexon is a small-molecule chemotherapeutic agent that has been shown to bind glutathione (GSH) and induce oxidative stress in tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, we investigate the effects of imexon on the integrated ER stress response in pancreatic carcinoma cells. Acute exposure to imexon induces an ER stress response characterized by accumulation of the oxidized form of the oxidoreductase Ero1α, phosphorylation of eIF2α, and inhibition of protein synthesis. An RNA interference chemosensitization screen identified the eukaryotic translation initiation factor eIF2B5 as a target that enhanced imexon-induced growth inhibition of MiaPaCa-2 pancreatic cancer cells, but did not significantly augment the effects of imexon on protein synthesis. Concurrent reduction of intracellular thiols with N-acetyl cysteine reversed imexon activity, however cotreatment with superoxide scavengers had no effect, suggesting thiol binding may be a primary component of the oxidative effects of imexon. Moreover, the data suggest that disruption of the redox balance in the ER is a potential therapeutic target.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hexanonas/farmacologia , Neoplasias Pancreáticas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Iniciação 2B em Eucariotos/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Oxirredução/efeitos dos fármacos , Oxirredutases/metabolismo , Neoplasias Pancreáticas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos
10.
Cancer Chemother Pharmacol ; 69(4): 1039-49, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22186884

RESUMO

PURPOSE: Preclinical studies evaluated the anti-tumor activity and mechanism of action of AMP423, a naphthyl derivative of 2-cyanoaziridine-1-carboxamide with structural similarity to the pro-oxidant anti-tumor agent imexon. METHODS: The cytotoxic potency was evaluated in vitro against a variety of human cancer cell lines. Mechanism-of-action studies were performed in the human 8226/S myeloma cell line and its imexon-resistant variant, 8226/IM10. In vivo activity was evaluated against human myeloma and lymphoma xenografts in SCID mice. Pharmacokinetics and toxicology were investigated in non-tumor-bearing mice. RESULTS: The 72-h IC(50)s for all cell types ranged from 2 to 36 µM, across a wide variety of human cancer cell lines. AMP423 was active in SCID mice bearing 8226/S myeloma and SU-DHL-6 B-cell lymphoma tumors, with a median tumor growth delay (T-C) of 21 days (P = 0.0002) and 5 days (P = 0.004), respectively, and a median tumor growth inhibition (T/C) of 33.3% (P = 0.03) and 82% (P = 0.01), respectively. In non-tumor-bearing mice, AMP423 was not myelosuppressive. Mechanistic studies show that AMP423's mode of cell death is a mixture of necrosis and apoptosis, with generation of reactive oxygen species, inhibition of protein synthesis, and a decrease in reduced sulfhydryl levels, but no alkylation of nucleophiles. Unlike its structural analog imexon, which causes cell cycle arrest in G(2)/M, AMP423 induces the accumulation of cells in S-phase. CONCLUSIONS: AMP423 has pro-oxidant effects similar to imexon, has greater cytotoxic potency in vitro, and has anti-tumor activity in hematologic tumors in vivo.


Assuntos
Antineoplásicos/farmacologia , Aziridinas/farmacologia , Linfoma de Células B/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Naftalenos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Aziridinas/química , Aziridinas/farmacocinética , Pontos de Checagem do Ciclo Celular , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Hexanonas/química , Hexanonas/farmacocinética , Hexanonas/farmacologia , Humanos , Linfoma de Células B/metabolismo , Masculino , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Naftalenos/química , Naftalenos/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Chemother Pharmacol ; 67(1): 183-92, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20339847

RESUMO

PURPOSE: Gemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo. METHODS: Median effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models. RESULTS: In three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM's pharmacokinetic disposition. In vitro, imexon did not alter GEM's metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM. CONCLUSIONS: These results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Área Sob a Curva , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Feminino , Hexanonas/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Invest New Drugs ; 27(1): 89-98, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18607542

RESUMO

Hypoxia-inducing factor-1 alpha (HIF-1alpha), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1alpha protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1alpha levels. Imexon did not reduce transcription of new HIF-1alpha mRNA, but did reduce the synthesis of new proteins, including HIF-1alpha, measured by (35)S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1alpha protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1alpha translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2alpha (eIF-2alpha) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug's cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1alpha, and this effect overcomes an increase in the stability of preformed HIF-1alpha due to proteasome inhibition by imexon. Because net HIF-1alpha levels are reduced by imexon, combination studies with other drugs affected by HIF-1alpha survival signaling are warranted.


Assuntos
Hexanonas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias Pancreáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
13.
Mol Cancer Ther ; 7(3): 500-9, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18319333

RESUMO

High-risk multiple myeloma can be correlated with amplification and overexpression of the cell cycle regulator CKS1B. Herein, we used the COMPARE algorithm to correlate high expression of CKS1B mRNA in the NCI-60 cell line panel with the concentration causing 50% growth inhibition (GI(50)) of >40,000 synthetic compounds. This led to the identification of NSC 338258 (EPED3), a highly stable, hydrophilic derivative of the plant alkaloid ellipticine. In vitro, this synthetic anticancer compound exhibits dramatic cytotoxic activity against myeloma cells grown in suspension or in coculture with stromal cells. EPED3-induced cell cycle arrest and an apoptotic progression that appear to be a consequence of the instantaneous effect of the drug on cytoplasmic organelles, particularly mitochondria. Disruption of mitochondria and cytoplasmic distribution of cytochrome c initiated the intracellular proteolytic cascade through the intrinsic apoptotic pathway. EPED3 is able to induce apoptosis in myeloma cells with de novo or acquired resistance to commonly administered antimyeloma agents. Collectively, our data suggest that EPED3 targets mitochondrial function to rapidly deplete chemical energy and initiate apoptosis in myeloma cells at nanomolar concentrations while leaving stromal cells unharmed.


Assuntos
Antineoplásicos/uso terapêutico , Elipticinas/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , CDC2-CDC28 Quinases , Proteínas de Transporte/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Quinases Ciclina-Dependentes/genética , Elipticinas/farmacologia , Citometria de Fluxo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia
14.
Biochem Pharmacol ; 75(4): 883-90, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18062937

RESUMO

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Although multiple myeloma patients often respond to initial therapy, the majority of patients will relapse with disease that is refractory to further drug treatment. Thus, new therapeutic strategies are needed. One common mechanism of acquired drug resistance involves a reduction in the expression or function of the drug target. We hypothesized that the cytotoxic activity of topoisomerase II (topo II) poisons could be enhanced, and drug resistance overcome, by increasing the expression and activity of the drug target, topo II in myeloma cells. To test this hypothesis, we evaluated the cytotoxicity of the anthracene-containing topo II poison, ethonafide (AMP-53/6-ethoxyazonafide), in combination with the proteasome inhibitor bortezomib (PS-341/Velcade). Combination drug activity studies were done in 8226/S myeloma cells and its drug resistant subclone, 8226/Dox1V. We found that a 24-h treatment of cells with bortezomib maximally increased topo IIalpha protein expression and activity, and consistently increased the cytotoxicity of ethonafide in the 8226/S and 8226/Dox1V cell lines. This increase in cytotoxicity corresponded to an increase in DNA double-strand breaks, as measured by the neutral comet assay. Therefore, increasing topo IIalpha expression through inhibition of proteasomal degradation increased DNA double-strand breaks and enhanced the cytotoxicity of the topo II poison ethonafide. These data suggest that bortezomib-mediated stabilization of topo IIalpha expression may potentiate the cytotoxic activity of topo II poisons and thereby, provide a strategy to circumvent drug resistance.


Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Isoquinolinas/farmacologia , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Inibidores da Topoisomerase II , Antígenos de Neoplasias , Bortezomib , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo II , Sinergismo Farmacológico , Humanos , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia
15.
J Pharmacol Exp Ther ; 321(3): 1109-17, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17351106

RESUMO

Ethonafide is an anthracene-containing derivative of amonafide that belongs to the azonafide series of anticancer agents. The lack of cross-resistance in multidrug-resistant cancer cell lines and the absence of a quinone and hydroquinone moiety make ethonafide a potentially less cardiotoxic replacement for existing anthracene-containing anticancer agents. For this study, we investigated the anticancer activity and mechanism of ethonafide in human prostate cancer cell lines. Ethonafide was cytotoxic against three human prostate cancer cell lines at nanomolar concentrations. Ethonafide was found to be better tolerated and more effective at inhibiting tumor growth compared with mitoxantrone in a human xenograft tumor regression mouse model. Mechanistically, we found that ethonafide inhibited topoisomerase II activity by stabilizing the enzyme-DNA complex, involving both topoisomerase IIalpha and -beta. In addition, ethonafide induced a potent G(2) cell cycle arrest in the DU 145 human prostate cancer cell line. By creating stable cell lines with decreased expression of topoisomerase IIalpha or -beta, we found that a decrease in topoisomerase IIalpha protein expression renders the cell line resistant to ethonafide. The decrease in sensitivity to ethonafide was associated with a decrease in DNA damage and an increase in DNA repair as measured by the neutral comet assay. These data demonstrate that ethonafide is a topoisomerase II poison and that it is topoisomerase IIalpha-specific in the DU 145 human prostate cancer cell line.


Assuntos
Isoquinolinas/farmacologia , Naftalimidas/farmacologia , Neoplasias da Próstata/prevenção & controle , Inibidores da Topoisomerase II , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/metabolismo , DNA de Cinetoplasto/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Docetaxel , Humanos , Isoquinolinas/uso terapêutico , Masculino , Melfalan/farmacologia , Camundongos , Camundongos SCID , Mitoxantrona/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica/efeitos dos fármacos , Quinoxalinas/farmacologia , Interferência de RNA , Taxoides/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cancer Chemother Pharmacol ; 59(6): 749-57, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17333195

RESUMO

PURPOSE: This study evaluated the cytotoxic effects of imexon (NSC-714597) in tumor cells when combined with a broad panel of chemotherapeutic drugs. METHODS: The sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays were used to analyze the degree of growth inhibition for the combination studies in the A375 human malignant melanoma and RPMI 8226 human multiple myeloma cell lines, respectively. Cells were continuously exposed to both drugs at a constant molar ratio for 4-5 days. Combination effects were analyzed using the Median Effect method. Statistical significance was inferred if the 95% confidence interval for the combination interaction (C.I.) values for a particular two-drug combination did not include 1.0 (additivity). Synergy was inferred for C.I. values<1.0 and antagonism for CI values>1.0. RESULTS: Imexon was synergistic when combined with DNA-binding agents (cisplatin, dacarbazine, melphalan) and pyrimidine-based antimetabolites (cytarabine, fluorouracil, gemcitabine) in both cell lines. Antagonistic combinations with imexon included methotrexate and the topoisomerase I (TOPO I) and II (TOPO II) inhibitors irinotecan, doxorubicin, mitoxantrone and etoposide. Docetaxel was synergistic with imexon in both cell lines whereas paclitaxel and fludarabine showed a mixed result. Dexamethasone and the proteasome inhibitor bortezomib showed synergy in myeloma cells and additivity in the melanoma cells. The vinca alkaloid, vinorelbine, and the multi-targeted antifol, pemetrexed, were additive with imexon in both cell lines. DISCUSSION: The consistent synergy seen for imexon and alkylating agents may relate to the sulfhydryl-lowering effect of imexon, which would render cells more sensitive to electrophilic species from the alkylators. The marked synergy noted with pyrimidine-based antimetabolites was unexpected and may relate to the induction of cell cycle arrest in S-phase. The strong antagonism noted for imexon with topoisomerase I and II inhibitors may be due to the effect of imexon at increasing oxidant levels which are known to antagonize the cytotoxic effects of topoisomerase poisons. In contrast, the synergy seen with bortezomib in myeloma cells may be related to an increase in reactive oxygen species (ROS) from both drugs. These results suggest that combinations of imexon with alkylating agents and pyrimidine-based antimetabolites are rational to pursue in therapeutic studies in vivo.


Assuntos
Hexanonas/administração & dosagem , Melanoma/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica , Ensaios de Seleção de Medicamentos Antitumorais , Hexanonas/uso terapêutico , Humanos , Células Tumorais Cultivadas
17.
Leuk Lymphoma ; 47(1): 97-109, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16321833

RESUMO

Imexon (NSC-714597) is an aziridine-containing imminopyrolidone in Phase I clinical trials. The current studies compared biological indices of cytotoxicity in 7 human multiple myeloma (MM) cell lines to develop a correlative model for imexon sensitivity. In the MM cell lines there was a wide range of sensitivity to imexon measured by standard cytotoxicity assays (MTT) and by viability/apoptosis/necrosis (Annexin-V-FITC/PI) measurements. The following sensitivity pattern was observed in order of decreasing sensitivity: IM-9 > 8226/S > MM.1S, ARH-77, H929 > 8226/I > U266. The same descending rank order was seen for loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and, at high drug concentrations, thiol depletion. Cell cycle analysis showed imexon sensitive cells accumulate at the G2/M interphase. Although there was a positive correlation between increasing CuZnSOD levels and imexon resistance, no relationship was found for catalase, Bcl-2, mitochondrial thioredoxin or MnSOD levels. These findings suggest consistent phenotypes for imexon sensitivity and resistance in human MM cell lines exposed to drug for 48 h, with a combination of apoptosis and necrosis. Resistance is correlated with CuZnSOD expression, reduced drug accumulation, lack of ROS generation and maintenance of MMP. Oxidation of cellular thiols occurs only at high (supra-cytotoxic) drug levels and is, therefore, weakly correlated with cytotoxicity. This unique mechanism involving oxidation and the previously reported absence of myelosuppression suggests that imexon may be rationally combined with existing cytotoxic agents to improve therapeutic activity in MM.


Assuntos
Hexanonas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/biossíntese , Glutationa/metabolismo , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sensibilidade e Especificidade , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismo , Células Tumorais Cultivadas
18.
Int J Gastrointest Cancer ; 36(1): 15-28, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16227632

RESUMO

Imexon is an aziridine-containing small molecule currently in Phase I clinical trials. This agent has been shown to bind to thiols and increase intracellular oxidants, inducing apoptosis in hematologic cancer cells. Pancreatic cancers are known to be sensitive to oxidation, suggesting this disease may be an appropriate target for this agent. The current report examines the activity of imexon in pancreatic cells. Imexon induced concentration-dependent and time-dependent apoptosis in a panel of six human pancreatic carcinoma cell (PCC) lines. The mean IC50 (SD) for growth inhibition by the SRB assay was 200 (101) microM for a 48 h exposure with a range of 64-358 microM. Cell killing was schedule-dependent, favoring exposure times > or =48 h. Imexon-treated MiaPaCa-2 cells underwent non-lethal growth arrest following exposure to concentrations < or =200 microM for 48 h. When concentrations were increased to 300 microM for > or =48 h, the MiaPaCa-2 cells arrested in G2 phase and activated caspases 3, 8, and 9 were detected. After a 72 h exposure to the IC80 concentration of imexon, cells exhibited a loss of mitochondrial membrane potential detected by CMXRos staining. However, there was no loss of reduced cellular thiols unless very high concentrations of > or =400 microM were used. In contrast, reactive oxygen species (ROS) were elevated in a dose-dependent fashion, starting at very low imexon concentrations. Imexon also significantly inhibited MiaPaCa-2 tumor growth in SCID mice at 100 mg/kg/d for 9 d. The tumor growth inhibition (% T/C) was 27% of control, and the tumor growth delay was 21 d, indicating an active agent by NCI standards. The levels of imexon that are cytotoxic in human PCC's are achievable based on the preliminary results of the ongoing Phase I trial. Imexon appears to be active against PCCs in vitro and has an entirely novel mechanism of action involving G2 arrest, accumulation of ROS, and the induction of apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Hexanonas/farmacologia , Neoplasias Pancreáticas/patologia , Relação Dose-Resposta a Droga , Humanos , Oxirredução , Espécies Reativas de Oxigênio , Células Tumorais Cultivadas
19.
Cancer Res ; 65(9): 3828-36, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15867381

RESUMO

The proteasome inhibitor bortezomib (also known as PS-341/Velcade) is a dipeptidyl boronic acid that has recently been approved for use in patients with multiple myeloma. Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, oligonucleotide microarray analysis of the 8226 multiple myeloma cell line showed a predominant induction of gene products associated with the endoplasmic reticulum secretory pathway following short-term, high-dose exposure to bortezomib. Examination of mediators of endoplasmic reticulum stress-induced cell death showed specific activation of caspase 12, as well as of caspases 8, 9, 7, and 3, and cleavage of bid. Treatment of myeloma cells with bortezomib also showed disregulation of intracellular Ca2+ as a mechanism of caspase activation. Cotreatment with a panel of Ca2+-modulating agents identified the mitochondrial uniporter as a critical regulatory factor in bortezomib cytotoxicity. The uniporter inhibitors ruthenium red and Ru360 prevented caspase activation and bid cleavage, and almost entirely inhibited bortezomib-induced cell death, but had no effect on any other chemotherapeutic drug examined. Additional Ca2+-modulating agents, including 2-amino-ethoxydiphenylborate, 1,2-bis (o-aminophenoxy) ethane-tretraacetic acid (acetoxymethyl) ester, and dantrolene, did not alter bortezomib cytotoxicity. Analysis of intracellular Ca2+ showed that the ruthenium-containing compounds inhibited Ca2+ store loading and abrogated the desensitized capacitative calcium influx associated with bortezomib treatment. These data support the hypothesis that intracellular Ca2+ disregulation is a critical determinant of bortezomib cytotoxicity.


Assuntos
Ácidos Borônicos/farmacologia , Cálcio/metabolismo , Mitocôndrias/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Pirazinas/farmacologia , Antineoplásicos/farmacologia , Bortezomib , Caspases/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas , Mitocôndrias/efeitos dos fármacos , Mieloma Múltiplo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Proteases/farmacologia
20.
Oncogene ; 22(47): 7396-402, 2003 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-14576847

RESUMO

The emergence of clinical drug resistance continues to be an obstacle for the successful treatment of cancer. Our current understanding of mechanisms associated with drug resistance has been ascertained by investigating drug-resistant models created by exposing a parental population to increasing concentrations of a cytotoxic. These unicellular drug-resistant models have been critical in elucidating drug-resistant mechanism and in some cases have aided in the identification of drug targets. However, these models do not address resistance mechanisms that contribute to de novo drug resistance. We propose that specific niches within the tumor microenvironment may provide a sanctuary for subpopulations of tumors cells that affords a survival advantage following initial drug exposure and may facilitate the acquisition of acquired drug resistance. More specifically, we propose that the bone marrow microenvironment is a sanctuary for hema-topoietic cancers. This review will focus on the bone marrow microenvironment and its role in conferring resistance to cytotoxics and physiological mediators of cell death.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Adesão Celular , Dano ao DNA/efeitos dos fármacos , Humanos , Integrinas/metabolismo , Transdução de Sinais
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