RESUMO
Activation of the mitogen-activated protein kinase (MAPK) cascade in mammalian cell lines positively regulates the G2/M transition. The molecular mechanism underlying this biological phenomenon remains poorly understood. Ribosomal S6 kinase (RSK) is a key downstream element of the MAPK cascade. Our previous studies established roles of RSK2 in Cdc25C activation during progesterone-induced meiotic maturation of Xenopus oocytes. In this study we demonstrate that both recombinant RSK and endogenous RSK in Xenopus egg extracts phosphorylate all three isoforms of human Cdc25 at a conserved motif near the catalytic domain. In human HEK293 and PC-3mm2 cell lines, RSK preferentially phosphorylates Cdc25A and Cdc25B in mitotic cells. Phosphorylation of the RSK sites in these Cdc25 isoforms increases their M-phase-inducing activities. Inhibition of RSK-mediated phosphorylation of Cdc25 inhibits G2/M transition. Moreover, RSK is likely to be more active in mitotic cells than in interphase cells, as evidenced by the phosphorylation status of T359/S363 in RSK. Together, these findings indicate that RSK promotes G2/M transition in mammalian cells through activating phosphorylation of Cdc25A and Cdc25B.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fosfatases cdc25/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Dados de Sequência Molecular , Oócitos/enzimologia , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Xenopus , Fosfatases cdc25/genéticaAssuntos
Derivados de Benzeno/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Piperazinas/farmacologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Benzamidas , Células Cultivadas , Dasatinibe , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Interleucina-3/farmacologia , Células K562 , Camundongos , Mutação/genética , Fosforilação/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tiazóis/farmacologia , Tirosina/metabolismoRESUMO
Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Benzenossulfonatos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/patologia , Mitocôndrias/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia , SorafenibeRESUMO
Monotherapy of chronic myeloid leukemia (CML) with imatinib mesylate has been cast into shadow by the evolution of clinical resistance during therapy. Resistance to imatinib can arise by multiple mechanisms including amplification or mutation of Bcr-Abl, and continuity of imatinib therapy is probably a poor option for either of these patient groups. Recently, however, a structurally distinct new class of drugs, the pyrido[2,3-d]pyrimidines, has been described, and these compounds are predicted to make different molecular contacts in the Abl kinase domain. These drugs potently target both the Bcr-Abl and Src-family kinase activities, both of which are thought to be relevant to survival of the leukemic cell. We asked whether these drugs could selectively induce cell death in murine cell line models of CML cells sensitive and resistant to imatinib by different mechanisms. We show that whereas the pyrido[2,3-d] pyrimidines are indeed highly potent in suppressing proliferation of Bcr-Abl-overexpressing imatinib-resistant cells, they are almost completely ineffective against cells expressing the T315I mutant. This implies that despite structural differences from imatinib, these drugs are unlikely to be useful in patients expressing this mutant Bcr-Abl protein, but may be effective in cases where selection of cells overexpressing the oncoprotein leads to refractoriness to imatinib.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/análise , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Benzamidas , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Camundongos , Pirimidinas/uso terapêutico , Resultado do Tratamento , Quinases da Família src/antagonistas & inibidoresRESUMO
beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Camptotecina/farmacologia , Caspases/metabolismo , Dicumarol/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Transformação Genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
The total syntheses of 12,13,15-desoxy-15(S)-aza-epothilone B (aza-dEpoB; dEpoB-lactam) and 12,13,15-desoxy-15(R)-aza-epothilone B (15-epi-aza-dEpoB; 15-epi-dEpoB-lactam) have been accomplished via a highly convergent strategy. We have also successfully oxidized 12,13,15-desoxy-15(S)-aza-epothilone B to aza-epothilone B (aza-EpoB; EpoB-lactam). Aza-epothilone B has been advanced to phase I clinical trials by the Bristol-Myers Squibb group. Our synthesis is efficient and was amenable to the production of significant quantities of these lactams. Using our fully synthetically derived lactams, in vitro and in vivo studies were conducted in comparison with advanced clinical candidates, 12,13-desoxyepothilone B and 12,13-desoxyepothilone F, also derived by total synthesis.
Assuntos
Antineoplásicos/síntese química , Epotilonas , Compostos de Epóxi/síntese química , Tiazóis/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Humanos , Células K562 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise Espectral , Tiazóis/química , Tiazóis/farmacologiaRESUMO
The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.
Assuntos
Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Vacinas Conjugadas/uso terapêutico , Adulto , Neoplasias Ósseas/secundário , Neoplasias Ósseas/terapia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Sequência de Carboidratos , Feminino , Humanos , Imunização , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Metástase Linfática , Pessoa de Meia-Idade , Dados de Sequência Molecular , Metástase Neoplásica , Resultado do Tratamento , Vacinas Conjugadas/efeitos adversos , Vacinas Conjugadas/imunologiaRESUMO
Earlier studies from this laboratory have shown that the uricosuric agent probenecid (PBCD) will inhibit the extrusion of folate analogues from tumor cells mediated by a plasma membrane ATPase resembling the canicular multispecific organic anion transporter/multidrug resistance-related protein (MRP) family of ATP binding cassette transporters. This inhibition of this outwardly directed membrane ATPase has been shown to have a favorable impact upon the cellular pharmacokinetics, cytotoxicity, and efficacy of methotrexate in vivo. In an extension of these earlier studies, which had focused only on murine ascites tumors, we now report that parental co-administration of PBCD will also enhance net intracellular accumulation in vitro and intracellular persistence in vivo of a new folate analogue, 10-propargyl-10-deazaaminopterin (PDX) in tumor cells. This resulted in marked enhancement of the efficacy of PDX against murine and human lung neoplasms and human prostate and mammary neoplasms growing as solid tumors in mice. As possible ATPases targeted by PBCD, all of these tumors expressed MRP-1, -4, and -7 genes, with expression of MRP-4 being greatest in each case. Four other MRP genes were expressed to a variable extent in some tumors but not others. The therapeutic enhancement of PDX by PBCD was manifested as tumor regression, where PDX alone was only growth inhibitory (A549 NSCL tumor), or as a substantial increase (3-4-fold) in overall regression and/or number of complete regressions (Lewis and LX-1 lung, PC-3 and TSU-PR1 prostate, and MX-1 mammary tumors) compared to PDX alone. Also, only in the case of PDX with PBCD, a significant number of mice transplanted with LX-1 or MX-1 tumors that experienced complete regression did not have regrowth of their tumor. In view of these results, clinical trials of this therapeutic modality appear to be warranted, especially in the case of new more efficacious folate analogues that are also permeants for this canicular multispecific organic anion transporter/MRP-like plasma membrane ATPase.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Aminopterina/análogos & derivados , Aminopterina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Probenecid/farmacologia , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Aminopterina/administração & dosagem , Aminopterina/farmacocinética , Animais , Proteínas de Transporte de Ânions , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Membrana Celular/enzimologia , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Antagonistas do Ácido Fólico/administração & dosagem , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/metabolismo , Probenecid/administração & dosagem , Probenecid/farmacocinética , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The efficacy of topoisomerase (Topo) I-active drugs may be improved by better understanding the molecular and cellular responses of tumor compared to normal cells after genotoxic insults. Ionizing radiation (IR) + Topo I-active drugs (e.g., Topotecan) caused synergistic cell killing in various human cancer cells, even in cells from highly radioresistant tumors. Topo I poisons had to be added either during or immediately after IR. Synergy was caused by DNA lesion modification mechanisms as well as by concomitant stimulation of two pathways of cell death: necrosis (IR) + apoptosis (Topo I poisons). Cumulative data favor a mechanism of synergistic cell killing caused by altered DNA lesion modification and enhanced apoptosis. However, alterations in cell cycle regulation may also play a role in the synergy between these two agents in certain human cancers. We recently showed that NF-kappa B, a known anti-apoptotic factor, was activated in various cancer cells after poisoning Topo I using clinically active drugs. NF-kappa B activation was dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. Cytoplasmic signaling leading to NF-kappa B activation after Topo I poisons was diminished in cytoplasts (lacking nuclei) and in CEM/C2 cells that expressed a mutant Topo I protein that did not interact with Topo I-active drugs. NF-kappa B activation was intensified in S-phase and blocked by aphidicolin, suggesting that activation was a result of double-strand break formation due to Topo I poisoning and DNA replication. Dominant-negative I kappa B expression augmented Topo I poison-mediated apoptosis. Elucidation of molecular signal transduction pathways after Topo I drug-IR combinations may lead to improved radiotherapy by blocking anti-apoptotic NF-kappa B responses. Recent data also indicate that synergy caused by IR + Topo I poisons is different from radiosensitization by beta-lapachone (beta-lap), a "reported" Topo I and II-alpha poison in vitro. In fact, beta-lap does not kill cells by poisoning either Topo I or II-alpha in vivo. Instead, the compound is "activated" by an IR (damage)-inducible enzyme, NAD(P)H:quinone oxidoreductase (NQO1), a gene cloned as x-ray-inducible transcript #3, xip3. Unlike the lesion modification pathway induced by IR + Topo I drugs, beta-lap kills cells via NQO1 futile cycle metabolism. Downstream apoptosis caused by beta-lap appears to be noncaspase-mediated, involving calpain or a calpain-like protease. Thus, although Topo I poisons or beta-lap in combination with IR both synergistically kill cancer cells, the mechanisms are very different.
Assuntos
Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Radiossensibilizantes/farmacologia , Inibidores da Topoisomerase I , Animais , Bovinos , DNA Topoisomerases Tipo I/fisiologia , Humanos , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The complex carbohydrate molecule globo H hexasaccharide has been synthesized, conjugated to keyhole limpet hemocyanin, and administered with the immunologic adjuvant QS-21 as a vaccine for patients with prostate cancer who have relapsed after primary therapies such as radiation or surgery. Globo H is one of several candidate antigens present on prostate cancer cells that can serve as targets for immune recognition and treatment strategies. The vaccine, given as five subcutaneous vaccinations over 26 weeks, has been shown to be safe and capable of inducing specific high-titer IgM antibodies against globo H. Its immunogenicity was confirmed in prostate cancer patients with a broad range of stages and tumor burdens. Observations of several patients who had evidence of disease relapse restricted to a rising biochemical marker, prostate-specific antigen (PSA), indicated that a treatment effect could occur within 3 months after completion of the vaccine therapy. This effect was manifested as a decline of the slope of the log of PSA concentration vs. time plot after treatment compared with values before treatment. Five patients continue to have stable PSA slope profiles in the absence of any radiographic evidence of disease for more than 2 years. The concept of using PSA slope profiles in assessing early treatment effects in biological therapies such as vaccines awaits further validation in phase II and III trials. The use of a variety of lesser known candidate glycoprotein and carbohydrate antigens in prostate cancer serves as a focus for the development of a multivalent vaccine of the treatment of relapsed prostate cancer in patients with minimal tumor burden.
Assuntos
Biomarcadores Tumorais/sangue , Vacinas Anticâncer/uso terapêutico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/terapia , Vacinas Conjugadas/uso terapêutico , Idoso , Anticorpos/sangue , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Sequência de Carboidratos , Proteínas do Sistema Complemento/imunologia , Testes Imunológicos de Citotoxicidade , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Seleção de Pacientes , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/prevenção & controle , Fatores de Tempo , Vacinação , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
We previously demonstrated that beta-lapachone (beta-lap) killed cancer cells solely by apoptosis. Beta-Lap induced apoptosis in HL-60 cells in a dose-dependent manner as measured by flow cytometry and DNA ladder formation. Cell cycle changes, such as accumulations in S and G2-phases, were not observed. Apoptosis was accompanied by activation of caspase 3 and concomitant cleavage of poly(ADP-ribose) polymerase (PARP) to an 89 kDa polypeptide. PARP cleavage was blocked by zDEVD-fmk or zVAD-fmk, caspase-specific cleavage site inhibitors. Retrovirally introduced bcl-2 prevented beta-lap-mediated caspase 3 activation and PARP cleavage and increased the viability of Bcl-2-expressing HL-60 cells compared to cells with vector alone. Various beta-lap-related analogs (e.g., dunnione and naphthoquinone derivatives) induced equivalent apoptosis in HL-60 cells, but no compound was more effective than beta-lap. These data provide further evidence that the primary mode of cell killing by beta-lap is by the initiation and execution of apoptosis in human cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Naftoquinonas/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Vetores Genéticos , Células HL-60/citologia , Células HL-60/enzimologia , Humanos , Naftoquinonas/farmacocinética , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
The quinazoline folate analogue raltitrexed (ZD1694; Tomudex) and the camptothecin derivative irinotecan are two new agents showing clinical activity against colorectal cancer. To identify the optimal conditions to achieve synergistic cytotoxicity before the clinical development of their combination, we explored the interactions between ZD1694 and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), in vitro. Cytotoxicity was evaluated with a clonogenic assay using the human colon cancer cell line HCT-8. Different schedules of administration and different dose ratios of the two agents were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay (T. C. Chou and P. Talalay, Adv. Enzyme Regul., 22: 27-55, 1984). Sequential short-term (1 and 4-h) exposures to SN-38 followed by ZD1694 resulted in synergistic cytotoxicity at broad dose-effect ranges. At a high level of cell kill, the synergism was greater when either equiactive doses of the two agents or higher relative doses of ZD1694 were used. A 24-h interval between exposure to SN-38 and ZD1694 significantly enhanced the magnitude of the synergy (P = 0.001). The opposite sequence or simultaneous exposures produced significantly less potentiation or nearly additive interactions (P = 0.0006 and P < 0.0001). The synergism was completely lost under conditions of more prolonged drug exposure (24-h continuous exposure). In conclusion, in this in vitro model of human colon cancer, ZD1694 and SN-38 produced synergistic cytotoxicity. Our findings support the clinical use of this combination and provide a rationale for a clinical trial using sequential short-term exposures to equiactive doses of SN-38 and ZD1694 administered sequentially with a 24-h interval.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Camptotecina/análogos & derivados , Neoplasias do Colo , Quinazolinas/farmacologia , Tiofenos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Sinergismo Farmacológico , Humanos , Irinotecano , Quinazolinas/administração & dosagem , Tiofenos/administração & dosagem , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Beta-lapachone (beta-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 microM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4-8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G1-S border. Apoptosis activated by beta-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 microM beta-lap. Topo I and Topo IIalpha levels decreased at > 24 h. Logarithmic-phase MCF-7 cells were not affected by < or = 1 microM beta-lap. In contrast, dramatic and irreversible G2-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 microM CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 microM) resulted in only approximately 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to > 5 microM CPT arrested at key cell cycle checkpoints (i.e., G1, S, and G2-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decreases in bcl-2, Topo I, Topo IIalpha, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed. Beta-lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Naftoquinonas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Dose Letal Mediana , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.
Assuntos
Aminopeptidases/metabolismo , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Metaloendopeptidases/metabolismo , Neovascularização Patológica/prevenção & controle , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Sequência de Aminoácidos , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/química , Animais , Sítios de Ligação , Bovinos , Cicloexanos , Humanos , Cinética , Mamíferos , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Metionil Aminopeptidases , Dados de Sequência Molecular , O-(Cloroacetilcarbamoil)fumagilol , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Oltipraz (5-pyrazinyl-4-methyl-1,2-dithiole-3-thione), which is undergoing clinical evaluation as an anticarcinogen, also inhibits HIV-1 replication (IC50 approximately equal to 10 microM). The inactivation of RT appears to be a relevant antiviral mechanism since oltipraz blocks viral replication in acutely infected T-cell lines, but is ineffective in chronically infected ACH-2 cells (H. J. Prochaska, W. G. Bornmann, P. Baron, and B. Polsky (1995) Mol. Pharmacol. 48, 15-20). Since a nucleophilic amino acid is a likely target for oltipraz, we assessed whether the conserved cysteine residues of HIV-1 RT (38Cys or 280Cys) were the target(s) for oltipraz, and we synthesized [Me 14C]oltipraz to determine if oltipraz forms a stable adduct with RT. Thus, HIV-2 RT as well as wild-type, 38Cys-->Ser, 280Cys-->Ser, and the Cys-->Ser double mutant of HIV-1 RT were purified from the lysates of transformed Escherichia coli strain DH5 alpha (A. Hizi, M. Shaharabany, R. Tal, and S. H. Hughes (1992) J. Biol. Chem. 267, 1293-1297) via a purification procedure that included (NH4)2SO4 fractionation followed by gel filtration, dye-ligand, and ion-exchange chromatographies. Procion yellow H4R was chosen as the dye-ligand chromatography since it was the most potent and selective inhibitor of RT among seventy reactive dyes that were screened. Mono Q anion-exchange chromatography with diethanolamine (pH 9) resulted in the generation of heterodimeric RT from a predominantly homodimeric enzyme preparation. Because the instability of dilute RT preparations at room temperature rendered the kinetic evaluation of inactivation difficult, we sought to identify conditions that prevent denaturation of these enzymes. High concentrations (25 mM) of MgCl2 had a stabilizing effect. Oltipraz behaved kinetically as an irreversible inhibitor of all RTs purified, and the kinetic constants for the inactivation of these enzymes were not significantly different from wild-type HIV-1 RT (Ki = 17.0 +/- 4.1 microM; k3 = 0.214 +/- 0.051 h-1). In stark contrast, oltipraz neither inhibited nor inactivated the Klenow fragment of DNA polymerase I, whose subdomain structure is similar to the p66 subunit of RT. Wild-type RT was incubated with 60 microM [Me 14C]oltipraz for 4 h and was then subjected to gel filtration chromatography. The [14C] label comigrated with RT with a stoichiometry of binding of 0.88 +/- 0.05 oltipraz per inactivated RT subunit (N = 3 experiments), and the [14C] label remained bound after treatment with 4 M urea.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antivirais/farmacologia , HIV-1/enzimologia , Pirazinas/farmacologia , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Antivirais/metabolismo , Sítios de Ligação , Cromatografia de Afinidade , DNA Polimerase I/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/genética , Transcriptase Reversa do HIV , HIV-1/genética , Cinética , Magnésio/farmacologia , Mutação , Pirazinas/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Inibidores da Transcriptase Reversa/metabolismo , Tionas , TiofenosRESUMO
Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication in vitro (ED50 approximately 10 microM), undergoes extensive metabolism in vivo. Most of the orally administered drug undergoes opening of the dithiolethione ring, reduction, recyclization, and methylation to form 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine ("metabolite III"). We report here that metabolite III inhibits viral replication in vitro (ED50 approximately 25 microM) in acutely infected H9 and CEM T cell lymphoma cell lines. Although both metabolite III and oltipraz were able to inhibit phorbol-12-myristate-13-acetate-stimulated viral replication in the chronically infected U1 promonocytic leukemia cell line, only metabolite III was able to inhibit phorbol-12-myristate-13-acetate-stimulated viral replication in chronically infected ACH-2 T cell lymphoma cells. The results with ACH-2 cells suggest that oltipraz inhibits an early stage of the viral life cycle, whereas metabolite III affects human immunodeficiency virus type 1 replication at a step distal to viral integration. This is consistent with the finding that oltipraz inhibits reverse transcriptase, whereas metabolite III does not. Although the mean ED50 for metabolite III in acutely infected peripheral blood mononuclear cells was 18 microM, the ED50 was below 5 microM in three of eight independent experiments. Studies of metabolite III in combination with oltipraz in acutely infected peripheral blood mononuclear cells demonstrated significant antiviral synergy. These results raise the possibility that the in vitro potency of oltipraz may underestimate its antiretroviral activity in vivo. Based on these results, the pharmacokinetics of oltipraz and metabolite III will be compared with the pharmacodynamic effects of orally administered oltipraz in a forthcoming phase I/II trial of oltipraz in patients with p24 antigenemia.
Assuntos
Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Pirazinas/metabolismo , Pirazinas/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Transcriptase Reversa do HIV , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Inibidores da Transcriptase Reversa , Tionas , Tiofenos , Células Tumorais CultivadasRESUMO
We have synthesized an analogue of vinblastine wherein a coumarin molecule is attached to the C17 of the vindoline moiety via a succinimide bridge (cou-VBL). cou-VBL exhibits fluorescence similar to that exhibited by coumarin. Chinese hamster ovary fibroblasts (LR73 cells) that exhibit an IC50 for vinblastine (VBL) of about 100 nM in growth inhibition assays are similarly sensitive to the cou-VBL compound. LR73 cells transfected with the mu MDR 3 gene that were subsequently selected on vinblastine (MDR35 cells) exhibit resistance to cou-VBL that is similar to their VBL resistance. A large change in the quantum efficiency of cou-VBL fluorescence accompanies efflux from intact cells, and comparison between cou-VBL and [3H]VBL efflux from the MDR35 cells reveals that the transport kinetics of the fluorescent analogue is very similar (if not identical) to that exhibited by [3H]VBL. Thus, similar to continuous monitoring of fluorescence (CMF) studies performed with the naturally fluorescent chemotherapeutic doxorubicin (Roepe, 1992), cou-VBL may be used in CMF studies aimed at rigorously defining the kinetics of VBL efflux from multidrug-resistant (MDR) cells. Initial data suggest the following: (1) A single exponential term approximates efflux from sensitive cells, whereas two exponentials are required to fit efflux from MDR35 cells. (2) The faster MDR35 term is virtually identical to the single term for the sensitive cells, whereas the other defines a process that is 5-20 times slower than passive diffusion, depending on the drug concentration. (3) The slower component has a much steeper and nearly linear dependence on drug concentration, whereas the fast passive diffusion component becomes asymptotic near 0.3 pM exchangeable drug/microgram of cell protein.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antineoplásicos/farmacocinética , Resistência a Múltiplos Medicamentos/fisiologia , Animais , Transporte Biológico/fisiologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cumarínicos/síntese química , Cumarínicos/farmacocinética , Cumarínicos/farmacologia , Cricetinae , Cricetulus , Análise dos Mínimos Quadrados , Vimblastina/análogos & derivados , Vimblastina/síntese química , Vimblastina/farmacocinética , Vimblastina/farmacologiaRESUMO
Bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha and interleukin-1 beta (IL-1 beta) stimulate similar cellular responses. TNF-alpha and IL-1 beta are known to initiate signaling through a pathway involving hydrolysis of sphingomyelin to ceramide (Kolesnick, R. N., and Golde, D. W. (1994) Cell 77, 325-328). In this system, ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies demonstrate that LPS, like TNF and IL-1, stimulates ceramide-activated protein kinase activity in human leukemia (HL-60) cells and in freshly isolated human neutrophils. Lipid A, the biologically active core of LPS, enhanced kinase activity in a time- and concentration-dependent manner. As little as 10 nM lipid A was effective, and a maximal effect occurred with 500 nM lipid A, increasing kinase activity 5-fold. Native LPS similarly induced kinase activation. This effect of LPS was markedly enhanced by LPS binding protein and required the LPS receptor CD14. In contrast to TNF and IL-1, LPS did not cause sphingomyelin hydrolysis and thus stimulates ceramide-activated protein kinase without generating ceramide. Molecular modeling showed strong structural similarity between ceramide and a region of lipid A. Based on these observations, we propose that LPS stimulates cells by mimicking the second messenger function of ceramide.
Assuntos
Ceramidas/química , Lipopolissacarídeos/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Configuração de Carboidratos , Células Cultivadas , Ceramidas/biossíntese , Ceramidas/metabolismo , Ativação Enzimática , Humanos , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-raf , Sistemas do Segundo Mensageiro , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Seven binary vinca alkaloid congeners were newly synthesized as the C14' or C16'(20') or C14'16'(20') stereoisomers of C20'-modified VBL. These congeners lacked detectable antimicrotubule activity in assays of polymerization of purified microtubule protein and of mitotic arrest induction. The compounds modulated the cytotoxicity of VBL, VCR, and DOX in sarcoma and colon-tumor cell lines. In wild-type cell lines, each congener elicited a concentration-dependent enhancement of cytotoxicity that was drug- and cell-type-selective. For example, C20'-deoxy C14'16'20'-epi VBL sensitized sarcoma S180 cells 19-fold to DOX and 11-fold to VCR but had no effect on VBL cytotoxicity. In the rat colon-cancer cell lines there was preferential enhancement of VCR cytotoxicity by most congeners. In two MDR cell strains of S180, the modulation potency of each congener was independent of specific drug or of resistance level. As a result, the amount of modulator (concentration) required for reversal was proportional to the drug-resistance level. Such properties were not displayed by the monomeric vinca alkaloid modulator vindoline. The potency of drug modulation in both wild-type and MDR cells strains was dependent on the stereoisomeric form of the congener and its C20'-substituents.