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 XenoenxertoRESUMO
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 fármacosRESUMO
Glucocorticoids are one component of combined treatment regimens for many types of lymphoma due to their ability to induce apoptosis in lymphoid cells. In WEHI7.2 murine thymic lymphoma cells, altering catalase and glutathione peroxidase activity by transfection or the use of chemical agents modulates the ability of glucocorticoids to induce apoptosis. This suggests that the oxidative stress response is important in determining the glucocorticoid sensitivity of the cells. For glutathione peroxidase and catalase to detoxify reactive oxygen species (ROS), reducing equivalents in the form of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) are ultimately required. The major source of NADPH in the cell is glucose 6-phosphate dehydrogenase (G6PDH). Therefore, we created G6PDH-overexpressing WEHI7.2 variants to test whether G6PDH activity is a key determinant of glucocorticoid sensitivity in WEHI7.2 cells. G6PDH-overexpressing WEHI7.2 cells were more sensitive to oxidative stress and glucocorticoids. The G6PDH-overexpressing WEHI7.2 variants appeared similar to cells undergoing glucose deprivation with decreased adenosine triphosphate (ATP) synthesis by the mitochondria and increased basal levels of ROS. Overexpression of G6PDH also sensitized the cells to other standard lymphoma chemotherapeutics including cyclophosphamide, doxorubicin, and vincristine. The decreased ATP and elevated ROS due to G6PDH overexpression may be key factors in increasing the sensitivity of the WEHI7.2 cells to lymphoma chemotherapeutics.
Assuntos
Apoptose , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Linfoma/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/análise , Caspases/metabolismo , Catalase/metabolismo , Linhagem Celular Tumoral , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Previsões , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Glucosefosfato Desidrogenase/análise , Glutationa/análise , Glutationa/metabolismo , Dissulfeto de Glutationa/análise , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Linfoma/tratamento farmacológico , Linfoma/patologia , Camundongos , NADP/análise , NADP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , TransfecçãoRESUMO
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 CultivadasRESUMO
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 , GencitabinaRESUMO
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 XenoenxertoRESUMO
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 fármacosRESUMO
Chronic inflammation increases lymphoma risk. Chronic inflammation exposes cells to increased reactive oxygen species (ROS). Constant exposure to ROS selects for oxidative stress-resistant cells with upregulated anti-oxidant defense enzymes. The impact of oxidative stress resistance on the redox biology and chemotherapy response in lymphoma has not been rigorously tested. To measure the effect of antioxidant defense enzyme upregulation in lymphoid cells, we created oxidative stress-resistant WEHI7.2 thymic lymphoma cell variants. We selected a population of WEHI7.2 cells for resistance to hydrogen peroxide and constructed catalase-overexpressing WEHI7.2 transfectants. The WEHI7.2 variants had: i) increased catalase and total superoxide dismutase activities; ii) an altered GSSG/2GSH redox potential; iii) a more oxidized NADP(+)/NADPH pool; and iv) increased phase 2 enzymes, NAD(P)H:quinone oxidoreductase and glutathione S-transferases µ and π. Regression analysis showed a correlation between the GSSG/2GSH redox potential and the increased phase 2 enzyme activities. As predicted from the anti-oxidant defense enzyme profile, the variants were more resistant to the oxidants hydrogen peroxide and paraquat. The variants exhibited resistance to the common lymphoma chemotherapeutics, cyclophosphamide, doxorubicin, vincristine and glucocorticoids. These data indicate that chronic ROS exposure results in lymphoid cells with multiple changes in their redox biology and a chemoresistance phenotype. These data further suggest that lymphomas that arise at the site of chronic inflammation develop chemoresistance due to a combination of drug detoxification and removal of ROS.
RESUMO
AIM: We evaluated mechanisms of interaction between the alkyating agent dacarbazine (DTIC) and the pro-oxidant, imexon, in the human A375 melanoma cell line. MATERIALS AND METHODS: The effect of DTIC and imexon, alone and in combination, was evaluated for growth inhibition (MTT), radiolabeled drug uptake, cellular thiol content (HPLC), and DNA strand breaks (Comet assay). Pharmacokinetic and antitumor effects were evaluated in mice. RESULTS: Growth inhibition in vitro was additive with the two drugs. There was no effect on drug uptake or on the number of DNA strand breaks. There was a >75% reduction in cellular glutathione and cysteine with imexon but not DTIC. Co-administration of the two drugs in mice caused an increase in the area under the curve of both drugs, but the combination was not effective in reducing human A375 melanoma tumors in vivo. CONCLUSION: Imexon and dacarbazine show additive effects in vitro but not in vivo in human A375 melanoma cells.
Assuntos
Antineoplásicos/farmacologia , Dacarbazina/farmacologia , Hexanonas/farmacologia , Melanoma/patologia , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Área Sob a Curva , Linhagem Celular Tumoral , Ensaio Cometa , Dacarbazina/sangue , Dacarbazina/farmacocinética , Interações Medicamentosas , Ensaios de Seleção de Medicamentos Antitumorais , Hexanonas/sangue , Hexanonas/farmacocinética , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCIDRESUMO
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 , GencitabinaRESUMO
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 , GencitabinaRESUMO
Imexon, a novel pro-oxidant, thiol-binding agent, is currently in phase I/II clinical trials in patients with advanced solid tumors. The aim of this study was to characterize the preclinical pharmacology of imexon in vivo. We investigated the anticancer activity of imexon in several cancer cell lines grown as xenografts in severe combined immunodeficient mice. Imexon was active against both hematologic and solid tumor types. The maximally tolerated dose, at the selected dosing schedule, was 150 mg/kg. Using the maximally tolerated dose of imexon, we sought to identify a potential pharmacodynamic biomarker to monitor the mechanistic effect systemically. As imexon binds cellular thiols in vitro, thiol depletion by imexon in vivo was evaluated as a potential biomarker. Following a single 150 mg/kg dose of imexon by intraperitoneal injection, glutathione levels decreased by 40% at 3 h in mouse erythrocytes. In mouse plasma, imexon treatment led to a significant decrease in cystine levels 2-4 h after drug administration. Notably, by this time, free imexon plasma levels were nondetectable. By investigating the pharmacokinetics of imexon, we also found that imexon undergoes rapid clearance from plasma in a dose-independent fashion with a half-life of 12-15 min. In summary, imexon is active against several cancer types in vivo. Imexon also decreases circulating thiols and exhibits dose-independent pharmacokinetics in mice. Plasma cystine levels may represent a biomarker of imexon activity in vivo.