RESUMO
Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Indazóis/farmacologia , Indóis/farmacologia , Venenos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Células HCT116 , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Indazóis/metabolismo , Indóis/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Venenos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/metabolismoRESUMO
Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771).
Assuntos
Antineoplásicos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Quinolonas/farmacologia , Proteína X Associada a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Farnesiltranstransferase/metabolismo , Humanos , Prenilação/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Células U937RESUMO
BACKGROUND: In preclinical studies the heat shock protein 90 (Hsp90) inhibitor tanespimycin induced down-regulation of checkpoint kinase 1 (Chk1) and other client proteins as well as increased sensitivity of acute leukemia cells to cytarabine. We report here the results of a phase I and pharmacological study of the cytarabine + tanespimycin combination in adults with recurrent or refractory acute leukemia. DESIGN AND METHODS: Patients received cytarabine 400 mg/m(2)/day continuously for 5 days and tanespimycin infusions at escalating doses on days 3 and 6. Marrow mononuclear cells harvested before therapy, immediately prior to tanespimycin, and 24 hours later were examined by immunoblotting for Hsp70 and multiple Hsp90 clients. RESULTS: Twenty-six patients were treated at five dose levels. The maximum tolerated dose was cytarabine 400 mg/m(2)/day for 5 days along with tanespimycin 300 mg/m(2) on days 3 and 6. Treatment-related adverse events included disseminated intravascular coagulation (grades 3 and 5), acute respiratory distress syndrome (grade 4), and myocardial infarction associated with prolonged exposure to tanespimycin and its active metabolite 17-aminogeldanamycin. Among 21 evaluable patients, there were two complete and four partial remissions. Elevations of Hsp70, a marker used to assess Hsp90 inhibition in other studies, were observed in more than 80% of samples harvested 24 hours after tanespimycin, but down-regulation of Chk1 and other Hsp90 client proteins was modest. CONCLUSIONS: Because exposure to potentially effective concentrations occurs only for a brief time in vivo, at clinically tolerable doses tanespimycin has little effect on resistance-mediating client proteins in relapsed leukemia and exhibits limited activity in combination with cytarabine. (Clinicaltrials.gov identifier: NCT00098423).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Leucemia/tratamento farmacológico , Doença Aguda , Adulto , Idoso , Benzoquinonas/administração & dosagem , Benzoquinonas/efeitos adversos , Quinase 1 do Ponto de Checagem , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/administração & dosagem , Lactamas Macrocíclicas/efeitos adversos , Leucemia/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Quinases/metabolismo , Recidiva , Fatores de TempoRESUMO
A phase 2 study of the oral farnesyltransferase inhibitor tipifarnib was conducted in 93 adult patients with relapsed or refractory lymphoma. Patients received tipifarnib 300 mg twice daily on days 1-21 of each 28-day cycle. The median number of prior therapies was 5 (range, 1-17). For the aggressive B-cell, indolent B-cell, and T-cell and Hodgkin lymphoma (HL/T) groups, the response rates were 17% (7/42), 7% (1/15), and 31% (11/36), respectively. Of the 19 responders, 7 were diffuse large B-cell non-Hodgkin lymphoma (NHL), 7 T-cell NHL, 1 follicular grade 2, and 4 HL. The median response duration for the 19 responders was 7.2 months (mean, 15.8 months; range, 1.8-62), and 5 patients in the HL/T group are still receiving treatment at 29-64+ months. The grade 3/4 toxicities observed were fatigue and reversible myelosuppression. Correlative studies suggest that Bim and Bcl-2 should be examined as potential predictors of response in future studies. These results indicate that tipifarnib has activity in lymphoma, particularly in heavily pretreated HL/T types, with little activity in follicular NHL. In view of its excellent toxicity profile and novel mechanism of action, further studies in combination with other agents appear warranted. This trial is registered at www.clinicaltrials.gov as #NCT00082888.
Assuntos
Linfoma/tratamento farmacológico , Quinolonas/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Relação Dose-Resposta a Droga , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Farnesiltranstransferase/antagonistas & inibidores , Feminino , Humanos , Linfoma/patologia , Masculino , Pessoa de Meia-Idade , Quinolonas/administração & dosagem , Quinolonas/efeitos adversos , Recidiva , Resultado do Tratamento , Adulto JovemRESUMO
Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/metabolismo , Carcinógenos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Ligante Fas/farmacologia , Células HL-60 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células Jurkat , Fosforilação/efeitos dos fármacos , Proteína Quinase C beta , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are receptor tyrosine kinases that participate in mitogenic and antiapoptotic signaling in normal and neoplastic epithelia. In the present study, immunoblotting and reverse transcription-PCR demonstrated expression of IGF1R and IR isoform A in acute myelogenous leukemia (AML) cell lines as well as in >80% of clinical AML isolates. Treatment with insulin enhanced signaling through the Akt and MEK1/2 pathways as well as survival of serum-starved AML cell lines. Conversely, treatment with BMS-536924, a dual IGF1R/IR kinase inhibitor that is undergoing preclinical testing, inhibited constitutive receptor phosphorylation as well as downstream signaling through MEK1/2 and Akt. These changes inhibited proliferation and, in some AML cell lines, induced apoptosis at submicromolar concentrations. Likewise, BMS-536924 inhibited leukemic colony formation in CD34+ clinical AML samples in vitro. Collectively, these results not only indicate that expression of IGF1R and IR isoform A is common in AML but also show that interruption of signaling from these receptors inhibits proliferation in clinical AML isolates. Accordingly, further investigation of IGF1R/IR axis as a potential therapeutic target in AML appears warranted.
Assuntos
Benzimidazóis/farmacologia , Leucemia Mieloide Aguda/enzimologia , Piridonas/farmacologia , Receptor IGF Tipo 1/biossíntese , Receptor de Insulina/biossíntese , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Células HL-60 , Humanos , Fator de Crescimento Insulin-Like I/biossíntese , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Isoformas de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Células U937RESUMO
The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results, we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age, 77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600, etoposide 100) and 8A (tipifarnib 400, etoposide 200). In vivo, tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as #NCT00112853.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Etoposídeo/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Quinolonas/administração & dosagem , Administração Oral , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Farnesiltranstransferase/antagonistas & inibidores , Células HL-60 , Humanos , Células Jurkat , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Células Tumorais Cultivadas , Células U937RESUMO
Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine (2',2'-difluoro 2'-deoxycytidine) and 7-ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of irinotecan), two S-phaseselective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony-forming assays revealed that simultaneous treatment of Ovcar-5 ovarian cancer cells or BxPC-3 pancreatic cancer cells with gemcitabine and SN-38 resulted in antagonistic effects. In contrast, sequential treatment with these two agents in either order resulted in synergistic anti-proliferative effects, although the mechanism of synergy varied with the sequence. In particular, SN-38 arrested cells in S phase, enhanced the accumulation of gemcitabine metabolites, and diminished checkpoint kinase 1, thereby sensitizing cells in the SN-38 --> gemcitabine sequence. Gemcitabine treatment followed by removal allowed prolonged progression through S phase, contributing to synergy of the gemcitabine --> SN-38 sequence. These results collectively suggest that S-phase-selective agents might exhibit more cytotoxicity when administered sequentially rather than simultaneously.
Assuntos
Camptotecina/análogos & derivados , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fase S/efeitos dos fármacos , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , DNA de Neoplasias/biossíntese , Desoxicitidina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Irinotecano , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ensaio Tumoral de Célula-Tronco , GencitabinaRESUMO
Human DNA topoisomerase I (topo I) is an essential mammalian enzyme that regulates DNA supercoiling during transcription and replication. In addition, topo I is specifically targeted by the anticancer compound camptothecin and its derivatives. Previous studies have indicated that topo I is a phosphoprotein and that phosphorylation stimulates its DNA relaxation activity. The locations of most topo I phosphorylation sites have not been identified, preventing a more detailed examination of this modification. To address this issue, mass spectrometry was used to identify four topo I residues that are phosphorylated in intact cells: Ser(10), Ser(21), Ser(112), and Ser(394). Immunoblotting using anti-phosphoepitope antibodies demonstrated that these sites are phosphorylated during mitosis. In vitro kinase assays demonstrated that Ser(10) can be phosphorylated by casein kinase II, Ser(21) can be phosphorylated by protein kinase Calpha, and Ser(112) and Ser(394) can be phosphorylated by Cdk1. When wild type topo I was pulled down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis demonstrated that Ser(21) phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity in vitro and interaction with DNA in cells.
Assuntos
Camptotecina , DNA Topoisomerases Tipo I/química , DNA/química , Regulação Enzimológica da Expressão Gênica , Mitose , Sequência de Aminoácidos , Proteína Quinase CDC2/metabolismo , Camptotecina/farmacologia , Humanos , Células K562 , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Homologia de Sequência de Aminoácidos , Serina/químicaRESUMO
Caspases are aspartate-directed cysteine proteases that cleave a diverse group of intracellular substrates to contribute to various manifestations of apoptosis. These proteases are synthesized as inactive precursors and are activated as a consequence of signaling induced by a wide range of physiological and pathological stimuli. Caspase activation can be detected by measurement of catalytic activity, immunoblotting for cleavage of their substrates, immunolabeling using conformation-sensitive antibodies or affinity labeling followed by flow cytometry or ligand blotting. Here we describe methods for each of these assays, identify recent improvements in these assays and outline the strengths and limitations of each approach.
Assuntos
Apoptose , Caspases/metabolismo , Marcadores de Afinidade , Animais , Galinhas , Ativação Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , ImmunoblottingRESUMO
Previous studies have suggested that Mcl-1, an antiapoptotic Bcl-2 homolog that does not exhibit appreciable affinity for the caspase 8-generated C-terminal Bid fragment (tBid), diminishes sensitivity to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). This study was performed to determine the mechanism by which Mcl-1 confers TRAIL resistance and to evaluate methods for overcoming this resistance. Affinity purification/immunoblotting assays using K562 human leukemia cells, which contain Mcl-1 and Bcl-x(L) as the predominant antiapoptotic Bcl-2 homologs, demonstrated that TRAIL treatment resulted in binding of tBid to Bcl-x(L) but not Mcl-1. In contrast, TRAIL caused increased binding between Mcl-1 and Bak that was diminished by treatment with the caspase 8 inhibitor N-(N(alpha)-acetylisoleucylglutamylthreonyl) aspartic acid (O-methyl ester)-fluoromethyl ketone (IETD(OMe)-fmk) or the c-Jun N-terminal kinase inhibitor SP600125. In addition, TRAIL caused increased binding of Bim and Puma to Mcl-1 that was inhibited by IETD(OMe)-fmk but not SP600125. Further experiments demonstrated that down-regulation of Mcl-1 by short hairpin RNA or the kinase inhibitor sorafenib increased TRAIL-induced Bak activation and death ligand-induced apoptosis in a wide variety of neoplastic cell lines as well as clinical acute myelogenous leukemia specimens. Collectively, these observations not only suggest a model in which Mcl-1 confers TRAIL resistance by serving as a buffer for Bak, Bim, and Puma, but also identify sorafenib as a potential modulator of TRAIL sensitivity.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Benzenossulfonatos/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Antineoplásicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Células HL-60 , Humanos , Células Jurkat , Células K562 , Proteína de Sequência 1 de Leucemia de Células Mieloides , Niacinamida/análogos & derivados , Compostos de Fenilureia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Sorafenibe , Proteína bcl-X/metabolismoRESUMO
Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.
Assuntos
Eosinófilos/química , Proteoglicanas/sangue , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Biomarcadores/sangue , Biomarcadores/química , Biomarcadores/urina , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/urina , Proteína Básica Maior de Eosinófilos , Eosinofilia/sangue , Fezes/química , Feminino , Humanos , Imunoensaio , Gravidez , Proteínas da Gravidez/sangue , Proteínas da Gravidez/urina , Proteoglicanas/imunologia , Proteoglicanas/isolamento & purificação , Proteoglicanas/urina , Homologia Estrutural de ProteínaRESUMO
The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.
Assuntos
Piperazinas/farmacologia , Piridonas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Fase G1/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Fase S/efeitos dos fármacosRESUMO
The BCR/ABL kinase has been targeted for the treatment of chronic myelogenous leukemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML and Ph+ acute lymphoblastic leukemia (ALL) are often resistant. In particular, mutation of the T315 residue in the bcr/abl activation loop renders cells highly resistant to imatinib and to second-generation kinase inhibitors such as BMS-354825 or AMN107. Adaphostin is a tyrphostin that was originally intended to inhibit the BCR/ABL kinase by competing with its peptide substrates. Recent findings have in addition implicated reactive oxygen species (ROS) in the cytotoxic mechanism of adaphostin. In view of this unique mode of action, we examined the effects of adaphostin on numerous imatinib-resistant leukemia models, including imatinib-resistant CML and Ph+ ALL cell lines, cells harboring point mutations in BCR/ABL, and specimens from imatinib-resistant CML patients, using assays for intracellular ROS, apoptosis, and clonogenicity. Every model of imatinib resistance examined remained fully sensitive to adaphostin-induced cell death. Collectively, these data suggest that ROS generation by adaphostin overcomes even the most potent imatinib resistance in CML and Ph+ ALL.
Assuntos
Adamantano/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Hidroquinonas/farmacologia , Mutação , Estresse Oxidativo/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Adamantano/farmacologia , Apoptose , Benzamidas , Linhagem Celular Tumoral , Células Clonais , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/patologia , Proteínas Tirosina Quinases/genética , Espécies Reativas de OxigênioRESUMO
Eosinophil granules contain several toxic cationic proteins that contribute to the pathophysiology of allergic diseases. These include eosinophil peroxidase, two ribonucleases, and two forms of the major basic protein (MBP). Extraction of eosinophil granules by exposure to acid solution and fractionation on Sephadex G-50 characteristically yields a distinctive profile of three discrete peaks, and these proteins are usually recovered in good quantities, except for the eosinophil major basic protein homolog (MBP2). We investigated the effect of multiple granule extractions by dilute HCl on the recovery of granule proteins. Isolated granules were repetitively extracted, up to 31 times, in 0.01 M HCl, and the extracts fractionated on Sephadex G-50. Whereas initial extracts yielded the characteristic three-peak fractionation pattern, later extracts yielded four discrete peaks. Characterization of the novel fourth peak showed that it contained MBP2. These results indicate that repetitive extraction of eosinophil granules yields an increased amount of all granule proteins, and that MBP2 can now be recovered in good quantities and in a relatively pure form.
Assuntos
Proteínas Granulares de Eosinófilos/isolamento & purificação , Eosinófilos/química , Proteínas Sanguíneas/isolamento & purificação , Fracionamento Celular , Separação Celular , Cromatografia em Gel , Grânulos Citoplasmáticos/química , Eletroforese em Gel de Poliacrilamida , Proteína Catiônica de Eosinófilo/isolamento & purificação , Proteína Básica Maior de Eosinófilos , Peroxidase de Eosinófilo/isolamento & purificação , Neurotoxina Derivada de Eosinófilo/isolamento & purificação , Eosinofilia/sangue , Eosinófilos/citologia , Flavinas/química , Humanos , Imunoensaio , Medições Luminescentes , Proteoglicanas/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
PURPOSE: To assess the maximum tolerated dose, toxicities, pharmacokinetics, and antileukemic activity of topotecan and carboplatin in adults with recurrent or refractory acute leukemias. EXPERIMENTAL DESIGN: Patients received topotecan and carboplatin by 5-day continuous infusion at nine dose levels. Patients achieving a complete remission received up to two additional courses for consolidation. Plasma topotecan and ultrafilterable platinum were assayed on days 1 to 5. In addition, pretreatment levels of various polypeptides in leukemic cells were examined by immunoblotting to assess possible correlations with response. RESULTS: Fifty-one patients received a total of 69 courses of therapy. Dose-limiting toxicity consisted of grade 4/5 typhlitis and grade 3/4 mucositis after one course of therapy or grade 4 neutropenia and thrombocytopenia lasting >50 days when a second course was administered on day 21. Among 45 evaluable patients, there were 7 complete remissions, 2 partial remissions, 1 incomplete complete remission, and 1 reversion to chronic-phase chronic myelogenous leukemia. Topotecan steady-state plasma concentrations increased with dose. No accumulation of topotecan or ultrafilterable platinum occurred between days 1 and 5 of therapy. Leukemic cell levels of topoisomerase I, checkpoint kinase 1, checkpoint kinase 2, and Mcl-1 correlated with proliferating cell nuclear antigen but not with response. In contrast, low Bcl-2 expression correlated with response (P = 0.014, Mann-Whitney U test). CONCLUSIONS: The maximum tolerated dose was 1.6 mg/m(2)/d topotecan plus 150 mg/m(2)/d carboplatin. The complete remission rate in a heavily pretreated population was 16% (33% at the highest three dose levels). Responses seem to correlate with low pretreatment blast cell Bcl-2 expression.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia/tratamento farmacológico , Doença Aguda , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Carboplatina/administração & dosagem , Carboplatina/efeitos adversos , Carboplatina/farmacocinética , Proteínas de Ciclo Celular/metabolismo , Terapia Combinada , DNA Topoisomerases Tipo I/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HL-60 , Transplante de Células-Tronco Hematopoéticas , Humanos , Immunoblotting , Infusões Intravenosas , Leucemia/metabolismo , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Topotecan/administração & dosagem , Topotecan/efeitos adversos , Topotecan/farmacocinética , Resultado do TratamentoRESUMO
Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.
Assuntos
Proteínas de Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular , Desoxicitidina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Citarabina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , GencitabinaRESUMO
Previous studies demonstrated that ataxia telangiectasia mutated- and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Rifabutina/análogos & derivados , Rifabutina/farmacologia , Benzoquinonas , Quinase 1 do Ponto de Checagem , Sinergismo Farmacológico , Células HL-60 , Humanos , Lactamas Macrocíclicas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno , Fase S/efeitos dos fármacosRESUMO
Agents that target topoisomerase I are widely utilized to treat human cancer. Previous studies have indicated that both the ataxia telangiectasia mutated (ATM)/checkpoint kinase (Chk) 2 and ATM- and Rad 3-related (ATR)/Chk1 checkpoint pathways are activated after treatment with these agents. The relative contributions of these two pathways to survival of cells after treatment with topoisomerase I poisons are currently unknown. To address this issue, we assessed the roles of ATR, Chk1, ATM, and Chk2 in cells treated with the topoisomerase I poisons camptothecin and 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan. Colony forming assays demonstrated that down-regulation of ATR or Chk1 sensitized cells to SN-38 and camptothecin. In contrast, ATM and Chk2 had minimal effect of sensitivity to SN-38 or camptothecin. Additional experiments demonstrated that the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin, which down-regulates Chk1, also sensitized a variety of human carcinoma cell lines to SN-38. Collectively, these results show that the ATR/Chk1 pathway plays a predominant role in the response to topoisomerase I inhibitors in carcinoma cells and identify a potential approach for enhancing the efficacy of these drugs.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Inibidores da Topoisomerase I , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Camptotecina/uso terapêutico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Deleção de Genes , Genes cdc , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19(+) purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.