RESUMO
UNLABELLED: Ionizing radiation (IR) is a key therapeutic regimen for many head and neck cancers (HNC). However, the 5-year overall survival rate for locally advanced HNCs is approximately 50% and better therapeutic efficacy is needed. NAD(P)H: quinone oxidoreductase 1 (NQO1) is overexpressed in many cancers, and ß-lapachone (ß-lap), a unique NQO1 bioactivatable drug, exploits this enzyme to release massive reactive oxygen species (ROS) that synergize with IR to kill by programmed necrosis. ß-Lap represents a novel therapeutic opportunity in HNC leading to tumor-selective lethality that will enhance the efficacy of IR. Immunohistochemical staining and Western blot assays were used to assess the expression levels of NQO1 in HNC cells and tumors. Forty-five percent of endogenous HNCs expressed elevated NQO1 levels. In addition, multiple HNC cell lines and tumors demonstrated elevated levels of NQO1 expression and activity and were tested for anticancer lethality and radiosensitization by ß-lap using long-term survival assays. The combination of nontoxic ß-lap doses and IR significantly enhanced NQO1-dependent tumor cell lethality, increased ROS, TUNEL-positive cells, DNA damage, NAD(+), and ATP consumption, and resulted in significant antitumor efficacy and prolonged survival in two xenograft murine HNC models, demonstrating ß-lap radiosensitization of HNCs through a NQO1-dependent mechanism. This translational study offers a potential biomarker-driven strategy using NQO1 expression to select tumors susceptible to ß-lap-induced radiosensitization. Mol Cancer Ther; 15(7); 1757-67. ©2016 AACR.
Assuntos
Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , NAD(P)H Desidrogenase (Quinona)/genética , Tolerância a Radiação/genética , Radiação Ionizante , Trifosfato de Adenosina/metabolismo , Animais , Catalase/genética , Catalase/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Modelos Animais de Doenças , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Radiossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Indole-3-carbinol (I3C) is a natural anti-carcinogenic compound found at high concentrations in Brassica vegetables. I3C was recently reported to inhibit neutrophil elastase (NE) activity, while consequently limiting the proteolytic processing of full length cyclin E into pro-tumorigenic low molecular weight cyclin E (LMW-E). In this study, we hypothesized that inhibition of NE activity and resultant LMW-E generation is critical to the anti-tumor effects of I3C. LMW-E was predominately expressed by ERα-negative breast cancer cell lines. However, ERα-positive cell lines demonstrated the greatest sensitivity to the anti-tumor effects of I3C and its more potent N-alkoxy derivatives. We found that I3C was incapable of inhibiting NE activity or the generation of LMW-E. Therefore, this pathway did not contribute to the anti-tumor activity of I3C. Gene expression analyzes identified ligand-activated aryl hydrocarbon receptor (AhR), which mediated sensitivity to the anti-tumor effects of I3C in ERα-positive MCF-7 cells. In this model system, the reactive oxygen species (ROS)-induced upregulation of ATF-3 and pro-apoptotic BH3-only proteins (e.g. NOXA) contributed to the sensitivity of ERα-positive breast cancer cells to the anti-tumor effects of I3C. Overexpression of ERα in MDA-MB-231 cells, which normally lack ERα expression, increased sensitivity to the anti-tumor effects of I3C, demonstrating a direct role for ERα in mediating the sensitivity of breast cancer cell lines to I3C. Our results suggest that ERα signaling amplified the pro-apoptotic effect of I3C-induced AhR signaling in luminal breast cancer cell lines, which was mediated in part through oxidative stress induced upregulation of ATF-3 and downstream BH3-only proteins.
Assuntos
Álcoois/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Indóis/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Álcoois/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina E/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/química , Elastase de Leucócito/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
AIMS: ß-Lapachone (ß-lap), a novel radiosensitizer with potent antitumor efficacy alone, selectively kills solid cancers that over-express NAD(P)H: quinone oxidoreductase 1 (NQO1). Since breast or other solid cancers have heterogeneous NQO1 expression, therapies that reduce the resistance (e.g., NQO1(low)) of tumor cells will have significant clinical advantages. We tested whether NQO1-proficient (NQO1(+)) cells generated sufficient hydrogen peroxide (H2O2) after ß-lap treatment to elicit bystander effects, DNA damage, and cell death in neighboring NQO1(low) cells. RESULTS: ß-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after ß-lap treatment. ß-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to ß-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (ß-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.
Assuntos
Efeito Espectador/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Quinonas/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Feminino , Humanos , Camundongos , Camundongos Nus , NAD(P)H Desidrogenase (Quinona)/deficiência , NAD(P)H Desidrogenase (Quinona)/genética , Oxirredução/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.
Assuntos
Antineoplásicos/química , Nitrilas/química , Piridinas/química , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cianoacrilatos , Dimerização , Humanos , Modelos Moleculares , Mieloma Múltiplo/tratamento farmacológico , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismoRESUMO
We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [(18)F]-labeled STI-571 was prepared with high specific activity (75 GBq/µmol) by nucleophilic displacement and an average radiochemical yield of 12%. [(131)I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [(18)F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [(18)F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.
Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Proteínas de Fusão bcr-abl/metabolismo , Piperazinas/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/uso terapêutico , Animais , Antineoplásicos/química , Benzamidas/química , Modelos Animais de Doenças , Humanos , Mesilato de Imatinib , Camundongos , Modelos Moleculares , Piperazinas/química , Pirimidinas/químicaRESUMO
A gram scale synthesis of the glucuronide metabolites of curcumin were completed in four steps. The newly synthesized curcumin glucuronide compounds 2 and 3 along with curcumin 1 were tested and their anti-proliferative effects against KBM-5, Jurkat cell, U266, and A549 cell lines were reported. Biological data revealed that as much as 1 µM curcumin 1 exhibited anticancer activity and almost 100% cell kill was noted at 10 µM on two out of four cell lines; while curcumin mono-glucuronide 2 as well as di-glucuronide 3 displayed no suppression of cell proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Curcumina/farmacologia , Linhagem Celular Tumoral , Curcumina/síntese química , Humanos , Células Jurkat , Relação Estrutura-AtividadeRESUMO
Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. ß-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of ß-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where â¼120 moles of superoxide were formed per mole of ß-lapachone in 2 minutes. ß-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of ß-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. ß-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of ß-lapachone and other NQO1 bioactivatable drugs.
Assuntos
Neoplasias da Mama/tratamento farmacológico , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/administração & dosagem , Poli(ADP-Ribose) Polimerases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catalase/genética , Catalase/metabolismo , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Necrose/genética , Necrose/patologia , Poli(ADP-Ribose) Polimerase-1 , Espécies Reativas de Oxigênio/metabolismoRESUMO
An improved method for the synthesis of 17ß-hydroxy-16α-iodo-wortmannin along with the first synthesis of 17ß-hydroxy-16α-iodoPX866 and [(131)I] radiolabeled 17ß-hydroxy-16α-[(131)I]iodo-wortmannin, as potential PET tracers for PI3K was also described. The differences between wortmannin and its iodo analogue were compared by covalently docking each structure to L833 in PI3K.
Assuntos
Androstadienos/química , Androstadienos/síntese química , Gonanos/síntese química , Compostos Radiofarmacêuticos/síntese química , Sítios de Ligação , Gonanos/química , Radioisótopos do Iodo/química , Marcação por Isótopo , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinase/química , Fosfatidilinositol 3-Quinase/metabolismo , Tomografia por Emissão de Pósitrons , Estrutura Terciária de Proteína , Compostos Radiofarmacêuticos/química , WortmaninaRESUMO
Curcumin (diferuloylmethane) is a potent anti-inflammatory and anti-tumorigenic agent that has shown preclinical activity in diverse cancers. Curcumin up-regulates heat shock protein 70 (hsp70) mRNA in several different cancer cell lines. Hsp70 contributes to an escape from the apoptotic effects of curcumin by several different mechanisms including prevention of the release of apoptosis inducing factor from the mitochondria and inhibition of caspases 3 and 9. Previously we showed that the combination of curcumin plus a heat shock protein inhibitor was synergistic in its down-regulation of the proliferation of a human schwannoma cell line (HEI-193) harboring an NF2 mutation, possibly because curcumin up-regulated hsp70, which also binds merlin, the NF2 gene product. In order to determine if curcumin also interacts directly with hsp70 and to discover other binding partners of curcumin, we synthesized biotinylated curcumin (bio-curcumin) and treated HEI-193 schwannoma cells. Cell lysates were prepared and incubated with avidin-coated beads. Peptides pulled down from this reaction were sequenced and it was determined that biotinylated curcumin bound hsp70, hsp90, 3-phosphoglycerate dehydrogenase, and a ß-actin variant. These binding partners may serve to further elucidate the underlying mechanisms of curcumin's actions.
Assuntos
Curcumina/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Fosfoglicerato Desidrogenase/química , Sítios de Ligação , Biotina/química , Linhagem Celular Tumoral , Curcumina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Simulação de Acoplamento Molecular , Neurilemoma/metabolismo , Neurilemoma/patologia , Fosfoglicerato Desidrogenase/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 µmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Carcinoma Ductal Pancreático/metabolismo , Cromolina Sódica/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromolina Sódica/análogos & derivados , Cromolina Sódica/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Ligação Proteica/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AG490 is a tyrosine kinase inhibitor with activity against Jak2 and apoptotic activity in specific leukemias. Due to its weak kinase inhibitory activity and poor pharmacology, we conducted a cell-based screen for derivatives with improved Jak2 inhibition and activity in animals. Two hits emerged from an initial small chemical library screen, and more detailed structure-activity relationship studies led to the development of WP1130 with 50-fold greater activity in suppressing Jak2-dependent cytokine signaling than AG490. However, WP1130 did not directly suppress Jak2 kinase activity, but mediated Jak2 ubiquitination resulting in its trafficking through HDAC6 to perinuclear aggresomes without cytokine stimulation or SOCS-1 induction. Jak2 primarily contained K63-linked ubiquitin polymers, and mutation of this lysine blocked Jak2 ubiquitination and mobilization in WP1130-treated cells. Further analysis demonstrated that WP1130, but not AG490, acts as a deubiquitinating enzyme (DUB) inhibitor, possibly through a Michael addition reaction. We conclude that chemical modification of AG490 resulted in development of a DUB inhibitor with activity against a DUB capable of modulating Jak2 ubiquitination, trafficking and signal transduction.
Assuntos
Endopeptidases/metabolismo , Janus Quinase 2/metabolismo , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Ubiquitinação , Substituição de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cianoacrilatos , Avaliação Pré-Clínica de Medicamentos , Endopeptidases/química , Ensaios Enzimáticos , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Concentração Inibidora 50 , Interleucina-6/farmacologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nitrilas/farmacologia , Fosforilação , Inibidores de Proteassoma , Transporte Proteico/efeitos dos fármacos , Proteólise , Piridinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
BACKGROUND: Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up-regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re-expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts. METHODS: Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5-aza-20-deoxycytidine [DAC] or 5-azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamicacid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re-expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate-binding Di-RAS-like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model. RESULTS: The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re-expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC-induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo. CONCLUSIONS: A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re-expression of imprinted tumor suppressor genes.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia , Azacitidina/análogos & derivados , Genes Supressores de Tumor/efeitos dos fármacos , Impressão Genômica , Ácidos Hidroxâmicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Decitabina , Sinergismo Farmacológico , Quimioterapia Combinada , Epigenômica , Feminino , Fase G2/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transplante Heterólogo , VorinostatRESUMO
AZD6244 is a small molecule inhibitor of the MEK (MAP/ERK kinase) pathway currently in clinical trials. However, the mechanisms mediating intrinsic resistance to MEK inhibition are not fully characterized. To define molecular mechanisms of MEK inhibitor resistance, we analyzed responses of 38 lung cancer cell lines following AZD6244 treatment and their genome-wide gene expression profiles and identified a panel of genes correlated with sensitivity or resistance to AZD6244 treatment. In particular, ingenuity pathway analysis revealed that activation of the STAT3 pathway was associated with MEK inhibitor resistance. Inhibition of this pathway by JSI-124, a STAT3-specific small molecule inhibitor, or with STAT3-specific siRNA sensitized lung cancer cells to AZD6244 and induced apoptosis. Moreover, combining a STAT3 inhibitor with AZD6244 induced expression of BIM and PARP cleavage, whereas activation of the STAT3 pathway inhibited BIM expression and elicited resistance to MEK inhibitors. We found that the STAT3-regulated microRNA miR-17 played a critical role in MEK inhibitor resistance, such that miR-17 inhibition sensitized resistant cells to AZD6244 by inducing BIM and PARP cleavage. Together, these results indicated that STAT3-mediated overexpression of miR-17 blocked BIM expression and caused resistance to AZD6244. Our findings suggest novel approaches to overcome resistance to MEK inhibitors by combining AZD6244 with STAT3 or miR-17 inhibitors.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Although chronic myelogenous leukemia (CML) is effectively controlled by Bcr-Abl kinase inhibitors, resistance to inhibitors, progressive disease, and incomplete eradication of Bcr-Abl-expressing cells are concerns for the long-term control and suppression of this disease. We describe a novel approach to targeting key proteins in CML cells with a ubiquitin-cycle inhibitor, WP1130. Bcr-Abl is rapidly modified with K63-linked ubiquitin polymers in WP1130-treated CML cells, resulting in its accumulation in aggresomes, where is it unable to conduct signal transduction. Induction of apoptosis because of aggresomal compartmentalization of Bcr-Abl was observed in both imatinib-sensitive and -resistant cells. WP1130, but not Bcr-Abl kinase inhibitors, directly inhibits Usp9x deubiquitinase activity, resulting in the down-regulation of the prosurvival protein Mcl-1 and facilitating apoptosis. These results demonstrate that ubiquitin-cycle inhibition represents a novel and effective approach to blocking Bcr-Abl kinase signaling and reducing Mcl-1 levels to engage CML cell apoptosis. This approach may be a therapeutic option for kinase inhibitor-resistant CML patients.
Assuntos
Apoptose , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Transdução de Sinais , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitinação , Apoptose/efeitos dos fármacos , Benzamidas , Linhagem Celular Tumoral , Cianoacrilatos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endopeptidases/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Mesilato de Imatinib , Modelos Biológicos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
PURPOSE: Pancreatic cancer is the fourth leading cause of cancer-related deaths, in which the 5-year survival rate is less than 5%. Current standard of care therapies offer little selectivity and high toxicity. Novel, tumor-selective approaches are desperately needed. Although prior work suggested that ß-lapachone (ß-lap) could be used for the treatment of pancreatic cancers, the lack of knowledge of the compound's mechanism of action prevented optimal use of this agent. EXPERIMENTAL DESIGN: We examined the role of NAD(P)H:quinone oxidoreductase-1 (NQO1) in ß-lap-mediated antitumor activity, using a series of MIA PaCa-2 pancreatic cancer clones varying in NQO1 levels by stable shRNA knockdown. The antitumor efficacy of ß-lap was determined using an optimal hydroxypropyl-ß-cyclodextran (HPß-CD) vehicle formulation in metastatic pancreatic cancer models. RESULTS: ß-Lap-mediated cell death required â¼90 enzymatic units of NQO1. Essential downstream mediators of lethality were as follows: (i) reactive oxygen species (ROS); (ii) single-strand DNA breaks induced by ROS; (iii) poly(ADP-ribose)polymerase-1 (PARP1) hyperactivation; (iv) dramatic NAD(+)/ATP depletion; and (v) programmed necrosis. We showed that 1 regimen of ß-lap therapy (5 treatments every other day) efficaciously regressed and reduced human pancreatic tumor burden and dramatically extended the survival of athymic mice, using metastatic pancreatic cancer models. CONCLUSIONS: Because NQO1 enzyme activities are easily measured and commonly overexpressed (i.e., >70%) in pancreatic cancers 5- to 10-fold above normal tissue, strategies using ß-lap to efficaciously treat pancreatic cancers are indicated. On the basis of optimal drug formulation and efficacious antitumor efficacy, such a therapy should be extremely safe and not accompanied with normal tissue toxicity or hemolytic anemia.
Assuntos
Antineoplásicos/uso terapêutico , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Naftoquinonas/uso terapêutico , Neoplasias Pancreáticas/enzimologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8â¶1, 4â¶1, 2â¶1, and 1â¶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8â¶1, 4â¶1, and 2â¶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1â¶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzimidazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Benzimidazóis/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent evidence suggests that several deubiquitinases (DUB) are overexpressed or activated in tumor cells and many contribute to the transformed phenotype. Agents with DUB inhibitory activity may therefore have therapeutic value. In this study, we describe the mechanism of action of WP1130, a small molecule derived from a compound with Janus-activated kinase 2 (JAK2) kinase inhibitory activity. WP1130 induces rapid accumulation of polyubiquitinated (K48/K63-linked) proteins into juxtanuclear aggresomes, without affecting 20S proteasome activity. WP1130 acts as a partly selective DUB inhibitor, directly inhibiting DUB activity of USP9x, USP5, USP14, and UCH37, which are known to regulate survival protein stability and 26S proteasome function. WP1130-mediated inhibition of tumor-activated DUBs results in downregulation of antiapoptotic and upregulation of proapoptotic proteins, such as MCL-1 and p53. Our results show that chemical modification of a previously described JAK2 inhibitor results in the unexpected discovery of a novel DUB inhibitor with a unique antitumor mechanism.
Assuntos
Apoptose/efeitos dos fármacos , Corpos de Inclusão/efeitos dos fármacos , Nitrilas/farmacologia , Piridinas/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Western Blotting , Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cianoacrilatos , Endopeptidases/genética , Endopeptidases/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Lung cancer is the leading cause of cancer-related deaths with current chemotherapies lacking adequate specificity and efficacy. Beta-lapachone (beta-lap) is a novel anticancer drug that is bioactivated by NAD(P)H:quinone oxidoreductase 1, an enzyme found specifically overexpressed in non-small cell lung cancer (NSCLC). Herein, we report a nanotherapeutic strategy that targets NSCLC tumors in two ways: (a) pharmacodynamically through the use of a bioactivatable agent, beta-lap, and (b) pharmacokinetically by using a biocompatible nanocarrier, polymeric micelles, to achieve drug stability, bioavailability, and targeted delivery. Beta-lap micelles produced by a film sonication technique were small ( approximately 30 nm), displayed core-shell architecture, and possessed favorable release kinetics. Pharmacokinetic analyses in mice bearing subcutaneous A549 lung tumors showed prolonged blood circulation (t(1/2), approximately 28 h) and increased accumulation in tumors. Antitumor efficacy analyses in mice bearing subcutaneous A549 lung tumors and orthotopic Lewis lung carcinoma models showed significant tumor growth delay and increased survival. In summary, we have established a clinically viable beta-lap nanomedicine platform with enhanced safety, pharmacokinetics, and antitumor efficacy for the specific treatment of NSCLC tumors.
Assuntos
Portadores de Fármacos/química , Neoplasias Pulmonares/tratamento farmacológico , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , Nanomedicina , Naftoquinonas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Micelas , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacocinética , Taxa de Sobrevida , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
(-)-Rhazinilam was spontaneously generated from a natural product during isolation. In cultured cells, it causes microtubule bundle formation, like those caused by paclitaxel. With tubulin, (-)-rhazinilam causes formation of an aberrant spiral polymer. Using glutamate and GTP, we developed an assay for spiral formation and applied it to 17 new (+/-)-rhazinilam analogs with either a modified side chain or a different size D ring. There was reasonable correlation between spiral formation and inhibition of human MCF-7 breast carcinoma cell growth. Only one side chain analog was as active as (+/-)-rhazinilam. During these studies, we observed that omitting GTP from the reaction mixture caused a major change in the morphology of the (-)-rhazinilam-induced polymer, with half the observed polymer being microtubule-like and half being spirals. This mixed polymer slowly disassembled at 0 degrees C, but there was no apparent difference in the lability of the microtubules versus the spirals.
Assuntos
Alcaloides/química , Alcaloides/farmacologia , Antimitóticos/química , Antimitóticos/farmacologia , Microtúbulos/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Alcaloides/síntese química , Animais , Antimitóticos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Centrifugação , Glutamatos/farmacologia , Guanosina Trifosfato/farmacologia , Humanos , Indolizinas/síntese química , Indolizinas/química , Indolizinas/farmacologia , Lactamas/síntese química , Lactamas/química , Lactamas/farmacologia , Ligação Proteica/efeitos dos fármacos , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
PURPOSE: Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved. EXPERIMENTAL DESIGN: Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models. RESULTS: PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy. CONCLUSIONS: In light of these findings, this novel molecular-based combination may be applicable to multiple STS histologic subtypes, and potentially merits rigorous evaluation in human STS clinical trials.