RESUMO
Cisplatin broadly functions as a routine treatment for lung adenocarcinoma (LUAD) patients. However, primary and acquired cisplatin resistances frequently occur in the treatment of LUAD patients, seriously affecting the therapeutic effect of cisplatin in patients. We intended to illustrate the impact of let-7c-5p/cell division cycle 25A (CDC25A) axis on cisplatin resistance in LUAD. Expression of let-7c-5p and CDC25A was analyzed via quantitative real-time polymerase chain reaction. The interaction between the two was verified by dual-luciferase reporter detection. For detecting half-maximal inhibitory concentration value of cisplatin in LUAD cells and cell proliferation, we separately applied Cell Counting Kit-8 and colony formation assays. Furthermore, we measured cell apoptosis and cell cycle distribution via flow cytometry, as well as cell cycle-related protein expression via Western blot. Let-7c-5p was evidently downregulated in LUAD, while CDC25A was remarkably upregulated. Let-7c-5p upregulation arrested LUAD cells to proliferate, stimulated cell apoptosis, and arrested cell cycle in G0/G1 phase, thus enhancing sensitivity of LUAD cells to cisplatin. In terms of mechanism, CDC25A was directly targeted by let-7c-5p, and the influence of let-7c-5p overexpression on LUAD proliferation, apoptosis, cell cycle, and cisplatin resistance could be reversed by CDC25A upregulation. Let-7c-5p improved sensitivity of LUAD cells to cisplatin by modulating CDC25A, and let-7c-5p/CDC25A axis was an underlying target for the intervention of LUAD cisplatin resistance.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , MicroRNAs , Humanos , Cisplatino/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Proliferação de Células , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Fosfatases cdc25/farmacologiaRESUMO
Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that are presently incurable. There have been reports of aberrant activation of cell cycle pathways in neurodegenerative diseases. Previously, we have found that Cdc25A is activated in models of neurodegenerative diseases, including AD and PD. In the present study, we have synthesized a small library of molecules targeting Cdc25A and tested their neuroprotective potential in cellular models of neurodegeneration. The Buchwald reaction and amide coupling were crucial steps in synthesizing the Cdc25A-targeting molecules. Several of these small-molecule inhibitors significantly prevented neuronal cell death induced by nerve growth factor (NGF) deprivation as well as 6-hydroxydopamine (6-OHDA) treatment. Lack of NGF signaling leads to neuron death during development and has been associated with AD pathogenesis. The NGF receptor TrkA has been reported to be downregulated at the early stages of AD, and its reduction is linked to cognitive failure. 6-OHDA, a PD mimic, is a highly oxidizable dopamine analogue that can be taken up by the dopamine transporters in catecholaminergic neurons and can induce cell death by reactive oxygen species (ROS) generation. Some of our newly synthesized molecules inhibit Cdc25A phosphatase activity, block loss of mitochondrial activity, and inhibit caspase-3 activation caused by NGF deprivation and 6-OHDA. Hence, it may be proposed that Cdc25A inhibition could be a therapeutic possibility for neurodegenerative diseases and these Cdc25A inhibitors could be effective treatments for AD and PD.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Doença de Parkinson , Humanos , Oxidopamina/toxicidade , Fator de Crescimento Neural/metabolismo , Fosfatases cdc25/metabolismo , Fosfatases cdc25/farmacologia , Dopamina/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/metabolismoRESUMO
RACGAP1 (Rac GTPase-activating protein 1) is correlated with tumor aggressiveness and poor prognosis, but the role of RACGAP1 in cervical cancer has not been fully reported. Analysis of RACGAP1 expression data in cervical cancer from the Cancer Genome Atlas (TCGA) database was carried out by GEPIA and UALCAN websites. In addition, the UALCAN database was used to identify the RACGAP1 positively correlated genes, which were used for the enrichment analysis. qRT-PCR, immunohistochemistry, western blot, and immunofluorescence were utilized to measure RACGAP1 expression in tissues and cells. Western blot, flow cytometry, MTT, and colony formation assays were applied to assess the effects of RACGAP1 on cell cycle, growth and viability in cervical cancer. Through bioinformatics analysis, we found that the level of RACGAP1 was aberrantly increased in cervical cancer, which was confirmed in cervical cancer tissues and cells. RACGAP1 associated genes, including CDC25C, were mainly enriched in cell cycle pathway, and RACGAP1 expression was negatively associated with CDC25C expression. RACGAP1 overexpression was related to patient's poor prognosis and promoted cervical cancer cell proliferation. Furthermore, RACGAP1 knockdown decreased the level of CDC2, p-CDC2, CDC25C, and Cyclin B1, inhibited proliferation and delayed cell cycle progression in cervical cancer cells. In mechanism, overexpression of CDC25C attenuated RACGAP1 knockdown-mediated cell growth inhibition and cell cycle arrest. Taken together, this study demonstrated that RACGAP1 was overexpressed in cervical cancer, and downregulation of RACGAP1 could inhibit the cervical cancer cell proliferation and cell cycle progression through regulating CDC25C expression.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Neoplasias do Colo do Útero , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Humanos , Neoplasias do Colo do Útero/genética , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Fosfatases cdc25/farmacologiaRESUMO
Background: Overexpression of nonmutated proteins involved in oncogenesis is a mechanism by which such proteins become immunogenic. We questioned whether overexpressed colorectal cancer associated proteins found at higher incidence and associated with poor prognosis could be effective vaccine antigens. We explored whether vaccines targeting these proteins could inhibit the development of intestinal tumors in the azoxymethane (AOM)-induced colon model and APC Min mice. Methods: Humoral immunity was evaluated by ELISA. Web-based algorithms identified putative Class II binding epitopes of the antigens. Peptide and protein specific T-cells were identified from human peripheral blood mononuclear cells using IFN-gamma ELISPOT. Peptides highly homologous between mouse and man were formulated into vaccines and tested for immunogenicity in mice and in vivo tumor challenge. Mice treated with AOM and APC Min transgenic mice were vaccinated and monitored for tumors. Results: Serum IgG for CDC25B, COX2, RCAS1, and FASCIN1 was significantly elevated in colorectal cancer patient sera compared to volunteers (CDC25B p=0.002, COX-2 p=0.001, FASCIN1 and RCAS1 p<0.0001). Epitopes predicted to bind to human class II MHC were identified for each protein and T-cells specific for both the peptides and corresponding recombinant protein were generated from human lymphocytes validating these proteins as human antigens. Some peptides were highly homologous between mouse and humans and after immunization, mice developed both peptide and protein specific IFN-γ-secreting cell responses to CDC25B, COX2 and RCAS1, but not FASCIN1. FVB/nJ mice immunized with CDC25B or COX2 peptides showed significant inhibition of growth of the syngeneic MC38 tumor compared to control (p<0.0001). RCAS1 peptide vaccination showed no anti-tumor effect. In the prophylactic setting, after immunization with CDC25B or COX2 peptides mice treated with AOM developed significantly fewer tumors as compared to controls (p<0.0002) with 50% of mice remaining tumor free in each antigen group. APC Min mice immunized with CDC25B or COX2 peptides developed fewer small bowel tumors as compared to controls (p=0.01 and p=0.02 respectively). Conclusions: Immunization with CDC25B and COX2 epitopes consistently suppressed tumor development in each model evaluated. These data lay the foundation for the development of multi-antigen vaccines for the treatment and prevention of colorectal cancer.
Assuntos
Antígenos de Neoplasias/farmacologia , Vacinas Anticâncer/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Ciclo-Oxigenase 2/farmacologia , Epitopos , Fosfatases cdc25/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Neoplasias/imunologia , Autoanticorpos/sangue , Vacinas Anticâncer/imunologia , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Ciclo-Oxigenase 2/imunologia , Feminino , Genes APC , Humanos , Imunidade Humoral , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Vacinação , Adulto Jovem , Fosfatases cdc25/imunologiaRESUMO
Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/fisiologia , Centrossomo/química , Quinases Ciclina-Dependentes/metabolismo , Mitose/efeitos dos fármacos , Fosfatases cdc25/metabolismo , Fosfatases cdc25/fisiologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/farmacologia , Linhagem Celular , Divisão do Núcleo Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Ciclina B1 , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/efeitos dos fármacos , Ativação Enzimática , Células HeLa , Humanos , Mitose/fisiologia , RNA Interferente Pequeno/farmacologia , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/farmacologiaRESUMO
Cell cycle G(2) checkpoint abrogation is an attractive strategy for sensitizing cancer cells to DNA-damaging anticancer agent without increasing adverse effects on normal cells. However, there is no single proven molecular target for this therapeutic approach. High-throughput screening for molecules inhibiting CHK1, a kinase that is essential for the G(2) checkpoint, has not yet yielded therapeutic G(2) checkpoint inhibitors, and the tumor suppressor phenotypes of ATM and CHK2 suggest they may not be ideal targets. Here, we optimized two G(2) checkpoint-abrogating peptides, TAT-S216 and TAT-S216A, based on their ability to reduce G(2) phase accumulation of DNA-damaged cells without affecting M phase accumulation of cells treated with a microtubule-disrupting compound. This approach yielded a peptide CBP501, which has a unique, focused activity against molecules that phosphorylate Ser(216) of CDC25C, including MAPKAP-K2, C-Tak1, and CHK1. CBP501 is >100-fold more potent than TAT-S216A and retains its selectivity for cancer cells. CBP501 is unusually stable, enters cells rapidly, and increases the cytotoxicity of DNA-damaging anticancer drugs against cancer cells without increasing adverse effects. These findings highlight the potency of CBP501 as a G(2)-abrogating drug candidate. This report also shows the usefulness of the cell cycle phenotype-based protocol for identifying G(2) checkpoint-abrogating compounds as well as the potential of peptide-based compounds as focused multitarget inhibitors.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Fase G2/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Fosfatases cdc25/metabolismo , Fosfatases cdc25/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Bleomicina/efeitos adversos , Bleomicina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Células HCT116 , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos SCID , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/patologia , Fragmentos de Peptídeos/química , Peptídeos/química , Fenótipo , Fosforilação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Fosfatases cdc25/químicaRESUMO
We have previously demonstrated that overexpression of Cdc25B in transgenic mice resulted in mammary gland hyperplasia and increased steroid hormone responsiveness. To address how Cdc25B enhances the hormone responsiveness in mammary glands, we showed that Cdc25B stimulates steroid receptor-dependent transcription in transient transfection assays and in a cell-free assay with chromatin templates. Surprisingly, the effect of Cdc25B on steroid receptors is independent of its protein phosphatase activity in vitro. The direct interactions of Cdc25B with steroid receptors, on the other hand, were evidenced in in vivo and in vitro assays, suggesting the potential direct contribution of Cdc25B on the steroid receptor-mediated transcription. In addition, p300/CBP-associated factor and CREB binding protein were shown to interact and synergize with Cdc25B and further enhance its coactivation activity. Thus, we have uncovered a novel function of Cdc25B that serves as a steroid receptor coactivator in addition to its role as a regulator for cell cycle progression. This dual function might likely contribute to its oncogenic action in breast cancer.
Assuntos
Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae , Fosfatases cdc25/metabolismo , Acetiltransferases/metabolismo , Animais , Proteína de Ligação a CREB , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/farmacologia , Linhagem Celular , Sistema Livre de Células/metabolismo , Ciclina D1/metabolismo , Ativação Enzimática/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Histona Acetiltransferases , Humanos , Proteínas Nucleares/metabolismo , Ratos , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Fosfatases cdc25/farmacologia , Fatores de Transcrição de p300-CBPRESUMO
Previous experiments from flight- and ground-based model systems indicate unexpected alterations of human leukocytes, leading to growth retardation and depression of mitogenic activation. The response of myelomonocytic U937 cells to simulated microgravity was therefore investigated. To this purpose, U937 cells were cultured in the NASA-developed bioreactor Rotating Wall Vessel (RWV) as a device to simulate microgravity on earth. No apoptosis was detected, in part because of the up-regulation of hsp70. In agreement with results obtained in space-flown U937 cells, the cells grew more slowly in the RWV than under normal conditions and this correlated with the down-modulation of cdc25B. Marked alterations of the cytokine secretion profile and, in particular, of inflammatory chemokines, as well as a decrease of the proteasome activity, were also observed in response to microgravity.
Assuntos
Ciclo Celular/fisiologia , Citocinas/metabolismo , Simulação de Ausência de Peso/efeitos adversos , Actinas/metabolismo , Western Blotting , Proteínas de Ciclo Celular/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Fatores Imunológicos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma , Células U937 , Fosfatases cdc25/farmacologiaRESUMO
Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of 'self-selection', and as a consequence exhibit diminished p16INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in fibroblast, p21Cip1 was not increased at senescence in HMECs. There was no increase in p27Kip1 levels nor in KIP association with targets cdks. While p15INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15INK4B. The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Senescência Celular , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Inibidores Enzimáticos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Supressoras de Tumor , Fosfatases cdc25/metabolismo , Adulto , Sequência de Aminoácidos , Mama/citologia , Proteínas de Transporte/metabolismo , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Quinase 2 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18 , Inibidor de Quinase Dependente de Ciclina p19 , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Proteínas Fúngicas/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares , Dados de Sequência Molecular , Inibidores de Proteínas Quinases , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais Cultivadas , Fosfatases cdc25/genética , Fosfatases cdc25/farmacologiaRESUMO
The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the effects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by caffeine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.
Assuntos
Toxinas Bacterianas/farmacologia , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Proteína Quinase CDC2/metabolismo , Cafeína/farmacologia , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Etoposídeo/farmacologia , Células HeLa/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Fase S/efeitos dos fármacos , Fosfatases cdc25/farmacologiaRESUMO
Bisperoxovanadium (bpV) compounds are irreversible protein tyrosine phosphatase (PTP) inhibitors with a spectrum of activity distinct from that of vanadium salts. We studied the efficacy of a panel of bpVs as antineoplastic agents in vitro and in vivo with a view to investigating phosphatases as potential antineoplastic targets. The Cdc25A dual-specificity phosphatase is an oncoprotein required for progression through G(1)-S. It cooperates with oncogenic Ras to transform cells and is overexpressed in several cancers. Cdc25A is therefore an attractive candidate phosphatase target for the antineoplastic activity of bpV compounds. Cytotoxicity was examined in 28 cancer cell lines and in vivo efficacy was examined in a DA3 murine mammary carcinoma model. In vitro phosphatase assays were used to directly measure phosphatase inhibition, comparing Cdc25A to hVH2/DSP4, leukocyte antigen related/receptor type PTPF catalytic domain (LAR), Yersinia pestis phosphatase (YOPH), and T-cell PTPase/non-receptor type PTP2 (TCPTP). CDK2 activity and Rb phosphorylation were examined by immunocomplex kinase assays and Western blot. Cdc25A is at least 20-fold more sensitive to bpV inhibition than hVH2/DSP4, and 3- to 10- fold more sensitive than TCPTP and LAR. bpV inhibition of Cdc25A in cells leads to CDK2 inactivation and hypophosphorylation Rb, resulting in G1-S arrest and induction of p53-independent apoptosis. The most cytotoxic analogue, bpV[4,7-dimethyl-1,10-phenanthroline-bisperoxo-oxo-vanadium (Me2Phen)], shows submicromolar IC50s against a panel of cell lines and inhibited tumor growth by 80% in mice. These results demonstrate that bpVs may have significant antineoplastic activity. In addition, they are in vitro and in vivo inhibitors of phosphatases including Cdc25A, suggesting that phosphatases may be appropriate targets for novel antineoplastic agents and that further development of these agents, targeting them to specific phosphatases such as CDC25A, may lead to novel agents with enhanced antineoplastic activity.
Assuntos
Antineoplásicos/farmacologia , Compostos de Vanádio/uso terapêutico , Fosfatases cdc25/farmacologia , Animais , Apoptose , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Fase G1 , Glutationa Transferase/metabolismo , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Modelos Químicos , Transplante de Neoplasias , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Fase SRESUMO
The CDC25 phosphatases regulate the cell division cycle by controlling the activity of cyclin-dependent kinases. While screening for inhibitors of phosphatases among natural products we repeatedly found that some polyprenyl-hydroquinones and polyprenyl-furans (furanoterpenoids) (furospongins, furospinosulins) were potent CDC25 phosphatase inhibitors. These compounds were extracted, isolated and identified independently from three sponge species (Spongia officinalis, Ircinia spinulosa, Ircinia muscarum), collected at different locations in the Mediterranean Sea. The compounds were inactive on the Ser/Thr phosphatase PP2C-alpha and on three kinases (CDK1, CDK5, GSK-3), suggesting that some potent and selective CDC25 phosphatase might be designed from these initial structures.
Assuntos
Ciclo Celular/efeitos dos fármacos , Furanos/farmacologia , Hidroquinonas/farmacologia , Poríferos/química , Fosfatases cdc25/efeitos dos fármacos , Fosfatases cdc25/farmacologia , Animais , Escherichia coli , Furanos/isolamento & purificação , Hidroquinonas/isolamento & purificaçãoRESUMO
CBP501 is an anticancer drug currently in randomized phase II clinical trials for patients with non-small cell lung cancer and malignant pleural mesothelioma. CBP501 was originally described as a unique G(2) checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, mitogen-activated protein kinase-activated protein kinase 2, and C-Tak1. However, unlike a G(2) checkpoint inhibitor, CBP501 clearly enhances the accumulation of tumor cells at G(2)-M phase that is induced by cisplatin or bleomycin at low doses and short exposure. By contrast, CBP501 does not similarly affect the accumulation of tumor cells at G(2)-M that is induced by radiation, doxorubicin, or 5-fluorouracil treatment. Our recent findings point to an additional mechanism of action for CBP501. The enhanced accumulation of tumor cells at G(2)-M upon combined treatment with cisplatin and CBP501 results from an increase in intracellular platinum concentrations, which leads to increased binding of platinum to DNA. The observed CBP501-enhanced platinum accumulation is negated in the presence of excess Ca(2+). Some calmodulin inhibitors behave similarly to, although less potently than, CBP501. Furthermore, analysis by surface plasmon resonance reveals a direct, high-affinity molecular interaction between CBP501 and CaM (K(d) = 4.62 × 10(-8) mol/L) that is reversed by Ca(2+), whereas the K(d) for the complex between CBP501 and 14-3-3 is approximately 10-fold weaker and is Ca(2+) independent. We conclude that CaM inhibition contributes to CBP501's activity in sensitizing cancer cells to cisplatin or bleomycin. This article presents an additional mechanism of action which might explain the clinical activity of the CBP501-cisplatin combination.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bleomicina/farmacologia , Calmodulina/metabolismo , Cisplatino/farmacologia , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Fosfatases cdc25/farmacologia , Bleomicina/administração & dosagem , Cloreto de Cálcio/farmacologia , Calmodulina/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacocinética , Adutos de DNA/biossíntese , Sinergismo Farmacológico , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Fragmentos de Peptídeos/farmacocinética , Fosfatases cdc25/farmacocinéticaRESUMO
In higher eukaryotes, the S phase and M phase of the cell cycle are triggered by different cyclin-dependent kinases (CDKs). For example, in frog egg extracts, Cdk1-cyclin B catalyzes entry into mitosis but cannot trigger DNA replication. Two hypotheses can explain this observation: Either Cdk1-cyclin B fails to recognize the key substrates of its S-phase-promoting counterparts, or its activity is somehow regulated to prevent it from activating DNA synthesis. Here, we show that Cdk1-cyclin B1 has cryptic S-phase-promoting abilities that can be unmasked by relocating it from the cytoplasm to the nucleus and moderately stimulating its activity. Subcellular localization of vertebrate CDKs and the control of their activity are thus critical factors for determining their specificity.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Replicação do DNA , Fase S , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/farmacologia , Extratos Celulares , Núcleo Celular/metabolismo , Cromatina/metabolismo , Ciclina B/farmacologia , Ciclina B1 , DNA/biossíntese , Ativação Enzimática , Mitose , Membrana Nuclear/metabolismo , Sinais de Localização Nuclear , Óvulo , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão , Transfecção , Xenopus , Fosfatases cdc25/metabolismo , Fosfatases cdc25/farmacologiaRESUMO
Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.