RESUMO
T-LAK cell originated protein kinase (TOPK) has been shown to regulate proliferation, invasion or migration of various cancer cells. However, the role of TOPK in follicle environments remains unknown. Here we reveal that TOPK inhibits TNF-α-induced human granulosa COV434 cell apoptosis. The expression of TOPK were increased in COV434 cells in response to TNF-α. TOPK inhibition also decreased TNF-α-induced SIRT1 expression but promoted TNF-α-induced p53 acetylation and expression of PUMA or NOXA. Accordingly, TOPK inhibition attenuated TNF-α-mediated SIRT1 transcriptional activity. In addition, SIRT1 inhibition augmented acetylation of p53 or expression of PUMA and NOXA in response to TNF-α, leading to COV434 cell apoptosis. We conclude that TOPK suppresses TNF-α-induced COV434 granulosa cell apoptosis via regulation of p53/SIRT1 axis, suggesting a potential role of TOPK in regulation of ovarian follicular development.
Assuntos
Apoptose , Células da Granulosa , Fator de Necrose Tumoral alfa , Proteína Supressora de Tumor p53 , Feminino , Humanos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células da Granulosa/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Hypoxia has been suggested to induce epithelial-mesenchymal transition (EMT) in various cancer types via the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). Here, we demonstrated that TOPK upregulates EMT and the invasion of H460 nonsmall-cell lung cancer cells through the induction of the HIF-1α/Snail axis and hypoxic signaling. The expression of endogenous TOPK, phosphorylated TOPK, HIF-1α and Snail was significantly increased upon hypoxia exposure, but TOPK depletion markedly abrogated the induced mRNA and protein levels of HIF-1α and Snail. Interestingly, TOPK knockdown restored the hypoxia-induced suppression of E-cadherin and diminished hypoxia-induced N-cadherin expression. In addition, Snail depletion suppressed hypoxia-induced N-cadherin expression, which was attenuated by TOPK knockdown. Moreover, knockdown of Snail decreased hypoxia-induced nonsmall-cell lung cancer cell migration and invasion, which were suppressed by TOPK depletion. In summary, we conclude that TOPK positively regulates HIF-1α expression through hypoxia signaling and thereby promotes Snail expression, leading to EMT and the invasion of nonsmall-cell lung cancer cells. These findings suggest that TOPK plays a critical role as a novel mediator of hypoxia signaling that regulates nonsmall-cell lung cancer development.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transição Epitelial-Mesenquimal , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Hipóxia TumoralRESUMO
TOPK has been suggested to contribute to invasion of lung, prostate, gastric, pancreatic or breast cancer cells. However, how TOPK mediates TGF-ß1/Smad signaling leading to epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells remains unknown. Here we report that TOPK upregulates T-box transcription factor TBX3 to enhance TGF-ß1-induced EMT and invasion of MDA-MB-231 breast cancer cells. Expression of endogenous TOPK was promoted by TGF-ß1 treatment of MDA-MB-231 cells time-dependently. In addition, knockdown of TOPK attenuated TGF-ß1-induced phosphorylation or transcriptional activity of Smad3. Meanwhile, levels of both mRNA and protein of TBX3 induced by TGF-ß1 were abolished by TOPK depletion. Also, knockdown of TBX3 inhibited TGF-ß1 induction of EMT-related genes Snail, Slug or Fibronectin. Furthermore, ablation of TOPK or TBX3 suppressed TGF-ß1-induced MDA-MB-231 cell invasion. Collectively, we conclude that TOPK positively regulates TBX3 in TGF-ß1/Smad signaling pathway, thereby enhancing EMT and invasion of breast cancer cells, implying a mechanistic role of TOPK in TGF-ß1/Smad signaling.
Assuntos
Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Smad/metabolismo , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais , Proteínas com Domínio T/metabolismo , Regulação para CimaRESUMO
TGF-ß1 is known to induce epithelial-mesenchymal transition (EMT), which is a prerequisite for cancer cell invasion. Here we reveal that TOPK upregulates EMT and invasion of human breast cancer MDA-MB-231 or Hs578T cells via NF-κB-dependent Snail/Slug in TGF-ß1 signaling. Endogenous TOPK expression was significantly increased in response to TGF-ß1 and TOPK knockdown mitigated TGF-ß1-induced breast cancer cell invasion. Interestingly, TOPK knockdown restored TGF-ß1 suppression of E-cadherin expression and markedly reduced N-cadherin induced by TGF-ß1. Also, NF-κB activity or expression of EMT markers Snail and Slug induced by TGF-ß1 was decreased by TOPK knockdown. Meanwhile, knockdown of Snail or TOPK attenuated TGF-ß1-induced breast cancer cell invasion. Taken, we conclude that TOPK mediates TGF-ß1-induced EMT and invasion in breast cancer cells via NF-κB/Snail signaling, suggesting novel role of TOPK as therapeutic target in TGF-ß1-mediated breast cancer development.
Assuntos
Neoplasias da Mama/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica/patologia , Fatores de Transcrição da Família Snail/genética , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Invasividade Neoplásica/genética , Transdução de Sinais , Regulação para CimaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia. The neuropathological features of AD include amyloid-ß (Aß) deposition and hyperphosphorylated tau accumulation. Although several clinical trials have been conducted to identify a cure for AD, no effective drug or treatment has been identified thus far. Recently, the potential use of non-pharmacological interventions to prevent or treat AD has gained attention. Low-dose ionizing radiation (LDIR) is a non-pharmacological intervention which is currently being evaluated in clinical trials for AD patients. However, the mechanisms underlying the therapeutic effects of LDIR therapy have not yet been established. In this study, we examined the effect of LDIR on Aß accumulation and Aß-mediated pathology. To investigate the short-term effects of low-moderate dose ionizing radiation (LMDIR), a total of 9 Gy (1.8 Gy per fraction for five times) were radiated to 4-month-old 5XFAD mice, an Aß-overexpressing transgenic mouse model of AD, and then sacrificed at 4 days after last exposure to LMDIR. Comparing sham-exposed and LMDIR-exposed 5XFAD mice indicated that short-term exposure to LMDIR did not affect Aß accumulation in the brain, but significantly ameliorated synaptic degeneration, neuronal loss, and neuroinflammation in the hippocampal formation and cerebral cortex. In addition, a direct neuroprotective effect was confirmed in SH-SY5Y neuronal cells treated with Aß1-42 (2 µM) after single irradiation (1 Gy). In BV-2 microglial cells exposed to Aß and/or LMDIR, LMDIR therapy significantly inhibited the production of pro-inflammatory molecules and activation of the nuclear factor-kappa B (NF-κB) pathway. These results indicate that LMDIR directly ameliorated neurodegeneration and neuroinflammation in vivo and in vitro. Collectively, our findings suggest that the therapeutic benefits of LMDIR in AD may be mediated by its neuroprotective and anti-inflammatory effects.
Assuntos
Doença de Alzheimer/radioterapia , Irradiação Craniana/métodos , Animais , Linhagem Celular Tumoral , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos da radiação , Feminino , Humanos , Camundongos , NF-kappa B/metabolismo , Doses de Radiação , Radiação IonizanteRESUMO
Background: Preterm birth is a known leading cause of neonatal mortality and morbidity. The underlying causes of pregnancy-associated complications are numerous, but infection and inflammation are the essential high-risk factors. However, there are no safe and effective preventive drugs that can be applied to pregnant women. Objective: The objectives of the study were to investigate a natural product, Abeliophyllum distichum leaf (ADL) extract, to examine the possibility of preventing preterm birth caused by inflammation. Methods: We used a mouse preterm birth model by intraperitoneally injecting lipopolysaccharides (LPS). ELISA, Western blot, real-time PCR and immunofluorescence staining analyses were performed to confirm the anti-inflammatory efficacy and related mechanisms of the ADL extracts. Cytotoxicity and cell death were measured using Cell Counting Kit-8 (CCK-8) analysis and flow cytometer. Results: A daily administration of ADL extract significantly reduced preterm birth, fetal loss, and fetal growth restriction after an intraperitoneal injection of LPS in mice. The ADL extract prevented the LPS-induced expression of TNF-α in maternal serum and amniotic fluid and attenuated the LPS-induced upregulation of placental proinflammatory genes, including IL-1ß, IL-6, IL-12p40, and TNF-α and the chemokine gene CXCL-1, CCL-2, CCL3, and CCL-4. LPS-treated THP-1 cell-conditioned medium accelerated trophoblast cell death, and TNF-α played an essential role in this effect. The ADL extract reduced LPS-treated THP-1 cell-conditioned medium-induced trophoblast cell death by inhibiting MAPKs and the NF-κB pathway in macrophages. ADL extract prevented exogenous TNF-α-induced increased trophoblast cell death and decreased cell viability. Conclusions: We have demonstrated that the inhibition of LPS-induced inflammation by ADL extract can prevent preterm birth, fetal loss, and fetal growth restriction.
Assuntos
Glucosídeos/química , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Oleaceae/química , Fenóis/química , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Nascimento Prematuro/induzido quimicamente , Nascimento Prematuro/prevenção & controle , Fator de Necrose Tumoral alfa/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Feminino , Masculino , Camundongos , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF), one of the two major TLRs-adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-ß in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células Epiteliais/metabolismo , Imunidade Inata/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Western Blotting , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Expressão Gênica/efeitos dos fármacos , Imunidade Inata/genética , Interferon beta/genética , Interferon beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Peritônio/citologia , Peritônio/metabolismo , Poli I-C/farmacologia , Pseudomonas aeruginosa/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genéticaRESUMO
T-lymphokine-activated killer cell-originated protein kinase (TOPK) is known to be up-regulated in cancer cells and appears to contribute to cancer cell proliferation and survival. However, the molecular mechanism by which TOPK regulates cancer cell survival still remains elusive. Here we show that TOPK directly interacted with and phosphorylated IκBα at Ser-32, leading to p65 nuclear translocation and NF-κB activation. We also revealed that doxorubicin promoted the interaction between nonphosphorylated or phosphorylated TOPK and IκBα and that TOPK-mediated IκBα phosphorylation was enhanced in response to doxorubicin. Also, exogenously overexpressed TOPK augmented transcriptional activity driven by either NF-κB or inhibitor of apoptosis protein 2 (cIAP2) promoters. On the other hand, NF-κB activity including IκBα phosphorylation and p65 nuclear translocation, as well as cIAP2 gene expression, was markedly diminished in TOPK knockdown HeLa cervical cancer cells. Moreover, doxorubicin-mediated apoptosis was noticeably increased in TOPK knockdown HeLa cells, compared with control cells, which resulted from caspase-dependent signaling pathways. These results demonstrate that TOPK is a molecular target of doxorubicin and mediates doxorubicin chemoresistance of HeLa cells, suggesting a novel mechanism for TOPK barrier of doxorubicin-mediated cervical cancer cell apoptosis.
Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfosserina/metabolismo , Neoplasias do Colo do Útero/enzimologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína 3 com Repetições IAP de Baculovírus , Células CHO , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Ubiquitina-Proteína Ligases , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
BACKGROUND: Cyclin D1 is immediately down-regulated in response to reactive oxygen species (ROS) and implicated in the induction of cell cycle arrest in G2 phase by an unknown mechanism. Either treatment with a protease inhibitor alone or expression of protease-resistant cyclin D1 T286A resulted in only a partial relief from the ROS-induced cell cycle arrest, indicating the presence of an additional control mechanism. METHODS: Cells were exposed to hydrogen peroxide (H2O2), and analyzed to assess the changes in cyclin D1 level and its effects on cell cycle processing by kinase assay, de novo synthesis, gene silencing, and polysomal analysis, etc. RESULTS: Exposure of cells to excessive H2O2 induced ubiquitin-dependent proteasomal degradation of cyclin D1, which was subsequently followed by translational repression. This dual control mechanism was found to contribute to the induction of cell cycle arrest in G2 phase under oxidative stress. Silencing of an eIF2α kinase PERK significantly retarded cyclin D1 depletion, and contributed largely to rescuing cells from G2 arrest. Also the cyclin D1 level was found to be correlated with Chk1 activity. CONCLUSIONS: In addition to an immediate removal of the pre-existing cyclin D1 under oxidative stress, the following translational repression appear to be required for ensuring full depletion of cyclin D1 and cell cycle arrest. Oxidative stress-induced cyclin D1 depletion is linked to the regulation of G2/M transit via the Chk1-Cdc2 DNA damage checkpoint pathway. GENERAL SIGNIFICANCE: The control of cyclin D1 is a gate keeping program to protect cells from severe oxidative damages.
Assuntos
Ciclo Celular/fisiologia , Ciclina D1/genética , Ciclina D1/metabolismo , Estresse Oxidativo/fisiologia , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Fase G2/genética , Fase G2/fisiologia , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Oxidative stress has been suggested to induce granulosa cell apoptosis, which contributes to follicular atresia. However, the mechanism via which oxidative stress mediates granulosa cell apoptosis remains elusive. Therefore, the aim of this study was to elucidate the molecular mechanisms regulating oxidative stressinduced granulosa cell apoptosis. The present study demonstrated that reactive oxygen species induced by H2O2 resulted in human granulosa COV434 cell apoptosis via the regulation of sirtuin 1 (SIRT1)mediated p53 activity. Endogenous SIRT1 expression was alleviated by H2O2 treatment of COV434 cells in a timedependent manner. In addition, knockdown or inhibition of SIRT1 promoted H2O2induced poly(ADPribose) polymerase (PARP) cleavage and p53 acetylation, which led to an increase in COV434 cell apoptosis. Treatment with H2O2 enhanced the expression levels of the p53dependent proteins, p53upregulated modulator of apoptosis (PUMA) and phorbol12myristate13acetateinduced protein 1 (PMAIP1), as well as those of p53; however, knockdown of p53 decreased cleaved PARP, PUMA and PMAIP1 expression levels induced by H2O2 treatment. Moreover, knockdown of PUMA or PMAIP1 attenuated the H2O2 induction of PARP cleavage and COV434 cell apoptosis. In conclusion, the present findings suggested that H2O2induced oxidative stress causes granulosa COV434 cell apoptosis via the upregulation of p53 activity by SIRT1 suppression, indicating a mechanistic role of the SIRT1/p53 axis in H2O2induced granulosa cell apoptosis.
Assuntos
Células da Granulosa/citologia , Peróxido de Hidrogênio/efeitos adversos , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Regulação para Baixo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/genética , Fatores de Tempo , Regulação para CimaRESUMO
T-lymphokine-activated killer cell-originated protein kinase (TOPK) appears to be highly expressed in various cancer cells and to play an important role in maintaining proliferation of cancer cells. However, the underlying mechanism by which TOPK regulates growth of cancer cells remains elusive. Here we report that upregulated endogenous TOPK augments resistance of cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). Stable knocking down of TOPK markedly increased TRAIL-mediated apoptosis of human HeLa cervical cancer cells, as compared with control cells. Caspase 8 or caspase 3 activities in response to TRAIL were greatly incremented in TOPK-depleted cells. Ablation of TOPK negatively regulated TRAIL-mediated NF-kappaB activity. Furthermore, expression of NF-kappaB-dependent genes, FLICE-inhibitory protein (FLIP), inhibitor of apoptosis protein 1 (c-IAP1), or X-linked inhibitor of apoptosis protein (XIAP) was reduced in TOPK-depleted cells. Collectively, these findings demonstrated that TOPK contributed to TRAIL resistance of cancer cells via NF-kappaB activity, suggesting that TOPK might be a potential molecular target for successful cancer therapy using TRAIL.
Assuntos
Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/genética , Caspase 3/genética , Caspase 8/genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêuticoRESUMO
Tau, a microtubule-associated protein expressed in mature neurons, interacts with tubulin to promote the assembly and stabilization of microtubules. However, abnormally hyperphosphorylated tau dissociates from microtubules and self-aggregates. Tau aggregates, including paired helical filaments and neurofibrillary tangles, promote neuronal dysfunction and death and are the defining neuropathological feature of tauopathies. Therefore, suppressing tau aggregation or stimulating the dissociation of tau aggregates has been proposed as an effective strategy for treating neurodegenerative diseases associated with tau pathology such as Alzheimer's disease (AD) and frontotemporal dementia. Interestingly, ginsenosides extracted from Panax ginseng reduced the hippocampal and cortical expression of phosphorylated tau in a rat model of AD. However, no studies have been conducted into the effect of red ginseng (RG) and its components on tau pathology. Here, we evaluated the effect of Korean red ginseng extract (KRGE) and its components on the aggregation and disassociation of tau. Using the thioflavin T assay, we monitored the change in fluorescence produced by the aggregation or disassociation of tau K18, an aggregation-prone fragment of tau441 containing the microtubule-binding domain. Our analysis revealed that KRGE not only inhibited tau aggregation but also promoted the dissociation of tau aggregates. In addition, the KRGE fractions, such as saponin, nonsaponin, and nonsaponin fraction with rich polysaccharide, also inhibited tau aggregation and promoted the dissociation of tau aggregates. Our observations suggest that RG could be a potential therapeutic agent for the treatment of neurodegenerative diseases associated with tauopathy.
Assuntos
Panax/química , Proteínas tau/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Humanos , RatosRESUMO
It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that Tlymphokineactivated killer celloriginated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stressinduced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa COV434 cell apoptosis induced by hydrogen peroxide (H2O2). Cotreatment with the TOPK inhibitor, OTS514, in combination with H2O2 increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H2O2induced COV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrinµ, diminished the augmentation in poly(ADPribose) polymerase (PARP) cleavage induced by OTS514 plus H2O2, while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H2O2, but promoted the H2O2induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53mediated granulosa cell apoptosis through SIRT1 downregulation in response to H2O2. Therefore, it can be concluded that TOPK suppresses H2O2induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.
Assuntos
Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Atresia Folicular/efeitos dos fármacos , Atresia Folicular/metabolismo , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
PDZ-binding kinase (PBK) has previously been shown to mediate chemoresistance of cancer cells to anticancer drugs. However, it remains unclear how PBK regulates paclitaxel-induced cancer cell death. Here, we demonstrate that PBK hinders paclitaxel-mediated autophagic cell death in H460 non-small-cell lung cancer cells. PBK knockdown increased apoptosis, autophagy, p53 level, and LC3 puncta upon paclitaxel treatment. Moreover, p53 expression facilitated an increase in the LC3-II/LC3-I ratio in response to paclitaxel, and PBK knockdown augmented paclitaxel-mediated p53 transcriptional activity. Meanwhile, paclitaxel induced PBK-mediated p53 nuclear export and its subsequent ubiquitination in control cells, but not in PBK knockdown cells. We conclude that PBK hampers paclitaxel-induced autophagic cell death by suppressing p53, suggesting a potential role of PBK in p53-mediated H460 cell death.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular Autofágica/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Paclitaxel/metabolismo , Paclitaxel/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Oncogenic activation of the mammalian target of rapamycin complex 1 (mTORC1) leads to endometrial cancer cell growth and proliferation. Sestrin2 (SESN2), a highly conserved stress-inducible protein, is involved in homeostatic regulation via inhibition of reactive oxygen species (ROS) and mTORC1. However, the role of SESN2 in human endometrial cancer remains to be investigated. Here, we investigated expression, clinical significance, and underlying mechanisms of SESN2 in endometrial cancer. SESN2 was upregulated more in endometrial cancer tissues than in normal endometrial tissues. Furthermore, upregulation of SESN2 statistically correlated with shorter overall survival and disease-free survival in patients with endometrial cancer. SESN2 expression strongly correlated with mTORC1 activity, suggesting its impact on prognosis in endometrial cancer. Additionally, knockdown of SESN2 promoted cell proliferation, migration, and ROS production in endometrial cancer cell lines HEC-1A and Ishikawa. Treatment of these cells with mTOR inhibitors reversed endometrial cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) marker expression. Moreover, in a xenograft nude mice model, endometrial cancer growth increased by SESN2 knockdown. Thus, our study provides evidence for the prognostic significance of SESN2, and a relationship between SESN2, the mTORC1 pathway, and endometrial cancer growth, suggesting SESN2 as a potential therapeutic target in endometrial cancer.
RESUMO
T-lymphokine-activated killer cell-originated protein kinase (TOPK) is overexpressed in highly proliferating tumors such as leukemias and myelomas, and seems to play a key role in tumorigenesis or metastasis. However, the precise role and regulatory mechanism explaining the effects of TOPK on tumor cells still remain elusive. Here, we reported that TOPK regulates UVB-induced c-Jun-NH2-kinase 1 (JNK1) activity, and is essential for H-Ras-induced activator protein-1 activity and cell transformation. We showed that TOPK associated with and phosphorylated JNK1 following UVB irradiation in vitro or in vivo. Moreover, UVB-induced JNK1 activity was greatly augmented in mouse epidermal JB6 Cl41 cells that stably expressed TOPK cDNA. On the other hand, JNK1 activity was markedly attenuated by stable expression of small interfering RNA against TOPK in malignant melanoma RPMI 7951 cells. Interestingly, TOPK interacted with JNK-interacting protein 1 and caused an elevation of JNK-interacting protein 1 scaffolding activity, thereby enhancing JNK1 activity. Furthermore, JNK1 was required for TOPK-mediated activator protein-1 transcriptional activity and transformed foci induced by UVB or H-Ras. Taken together, these findings showed that TOPK positively modulated UVB-induced JNK1 activity and played a pivotal role in JNK1-mediated cell transformation induced by H-Ras. These studies might also provide a novel molecular mechanism for the role of TOPK in UVB-mediated skin carcinogenesis.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Genes ras , Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , DNA Complementar/genética , Ativação Enzimática/efeitos da radiação , Humanos , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Células Swiss 3T3 , Fator de Transcrição AP-1/metabolismo , Transfecção , Raios UltravioletaRESUMO
Resveratrol, present in grapes and red wine, is reported to be a natural chemopreventive agent against cancer. However, the concentrations required to exert these effects may be difficult to achieve by drinking only 1 or 2 glasses of red wine a day. Therefore, developing more potent, nontoxic analogues of resveratrol may provide a feasible means of achieving an effective physiologic concentration. Here we report that the resveratrol analogue, 3,5,3',4',5'-pentahydroxy-trans-stilbene (RSVL2), inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation in JB6 P+ mouse epidermal cells. Further, we identified MEK/ERK signaling as the direct molecular target for the anticancer effects of RSVL2 and demonstrated that RSVL2 inhibited MEK1, but not Raf1 or ERK2 kinase activity. RSVL2 also dose-dependently suppressed MEK1 kinase activity induced by TPA and the inhibition of H-Ras-induced cell transformation was much stronger for RSVL2 than for PD098059 or resveratrol. Both in vitro and ex vivo pull-down assays indicated that RSVL2, but not resveratrol, directly bound with GST-MEK1, but did not compete with ATP for binding. Docking data indicated that the low inhibitory activity of resveratrol might be due to the lack of the hydroxyl group at the meta position of the B ring, thereby preventing resveratrol from forming a hydrogen bond with the backbone amide group of Ser212, which is the key interaction for stabilizing the inactive conformation of the activation loop.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estilbenos/química , Estilbenos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Modelos Moleculares , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Resveratrol , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Inflammation has been known to be linked to invasion or metastasis of breast cancer, which has poor prognosis, although the regulatory mechanism remains to be undiscovered. Here we show that T-LAK cell-originated protein kinase (TOPK) mediates pro-inflammatory endotoxin lipopolysaccharide (LPS)-induced breast cancer cell migration and invasion. The mRNA or protein level of TOPK, toll- like receptor4 (TLR4), interleukin (IL)-6, vascular endothelial growth factor (VEGF) or matrix metalloproteinase9 (MMP9) genes related to TLR4 signaling or tumor progression was induced by LPS treatment in MCF7 breast cancer cells, but the induction was abolished by stable knocking down of TOPK in MCF7 cells. Also, TOPK depletion decreased LPS-induced phosphorylation of p38, but not ERK and JNK among mitogen-activated protein kinases (MAPKs). On the other hand, we revealed that TOPK is essential for transcriptional activity of NF-κB or MMP9 promoter triggered by LPS. The induced promoter activity of NF-κB or MMP9 but not AP-1 was inhibited by knocking down of TOPK. Furthermore, we demonstrated that inhibitor of TOPK or MMP9 as well as MMP9 siRNA efficiently blocked LPS-induced migration or invasion of breast cancer cell lines. Interestingly, both of expression of TOPK and TLR4 were markedly increased in high-grade breast cancer. Collectively, we conclude that TOPK functions as a key mediator of LPS/TLR4-induced breast cancer cell migration and invasion through regulation of MMP9 expression or activity, implying a potential role of TOPK as a therapeutic target linking LPS-induced inflammation to breast cancer development.
Assuntos
Neoplasias da Mama/genética , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Lipopolissacarídeos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Adulto , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Tuberculosis due to Mycobacterium tuberculosis infection is a leading cause of death worldwide. Recognition of this pathogen is crucial for the activation of innate and adaptive immune responses. Nucleotide-binding oligomerization domain (Nod)1 and Nod2 are cytoplasmic receptors that can detect unique muropeptides of bacterial peptidoglycan. Nod2 is critical for the initiation of the host immune response against M. tuberculosis infection, however the role of Nod1 remains largely unknown. We investigated the role of Nod1 with respect to cytokine production by bone marrow-derived macrophages (BMDMs) in response to M. tuberculosis infection. Production of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1ß were induced in BMDMs; cytokine levels were not affected by a deficiency in Nod1. Activation of NF-κB and MAPKs was also comparable between wild-type and Nod1-deficient BMDMs. Levels of IL-6 and IL-1ß were reduced in Nod1/Nod2 double-deficient BMDMs to a greater extent than in Nod2-deficient cells. Furthermore, when signaling of Toll-like receptors (TLRs) was inhibited by lipopolysaccharide pre-treatment, cytokine production was diminished in Nod1-deficient BMDMs. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis infection.
Assuntos
Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Receptores Toll-Like/imunologia , Animais , Regulação da Expressão Gênica , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Cultura Primária de Células , Transdução de Sinais , Receptores Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Although a Yersinia pseudotuberculosis (Yptb) lung infection model has been developed to study Y. pestis pathogenesis, it is still necessary to establish a new animal model to mimic the pathophysiological features induced by Y. pestis infection. Here, we provide a new lung infection model using the Yptb strain, IP2777, which displayed rapid spread of bacteria to the liver, spleen, and blood. In addition, we examined whether TLR4 is involved in Yptb-induced pathogenesis in the lung infection model of mice we generated. Following lung infection of WT and TLR4-deficient mice with the Yptb strain IP2777, the survival rate, bacterial colonization, histopathology, and level of cytokines and chemokines in the lung, spleen, liver, and blood were analyzed. TLR4-deficient mice had a lower survival rate than WT mice in response to Yptb lung infection. Although the bacterial colonization and pathology of the lung were comparable between WT and TLR4-deficient mice, those of the spleen and liver were more severe in TLR4-deficient mice. In addition, the levels of TNF-α and CXCL2 in the liver and IL-6 and CXCL2 in the blood were higher in TLR4-deficient mice than in WT mice. Our results demonstrate that TLR4 is necessary for optimal host protection against Yptb lung infection and TLR4-deficient mice may serve as a better genetic model of Yptb infection for mimicking Y. pestis infection.