RESUMO
Breast cancer is one of the major malignancies in women, and most related deaths are due to recurrence, drug resistance, and metastasis. The expression of the mouse double minute 2 (MDM2) oncogene is upregulated in breast cancer; however, its regulatory mechanism has yet to be fully elucidated. Herein, we identified the tumor suppressor death-associated protein kinase 1 (DAPK1) as a novel MDM2 regulator by unbiased peptide library screening. DAPK1 is directly bound to MDM2 and phosphorylates it at Thr419. DAPK1-mediated MDM2 phosphorylation promoted its protein degradation via the ubiquitin-proteasome pathway, resulting in upregulated p53 expression. DAPK1 overexpression, but not its kinase activity-deficient form, decreased colony formation and increased doxorubicin-induced cell death; however, DAPK1 knockdown produced the opposite effects in human breast cancer cells. In a xenograft tumorigenesis assay, DAPK1 overexpression significantly reduced tumor formation, whereas inhibition of DAPK1 kinase activity reduced its antitumorigenic effect. Finally, DAPK1 expression was negatively correlated with MDM2 levels in human breast cancer tissues. Thus, these results suggest that DAPK1-mediated MDM2 phosphorylation and its protein degradation may contribute to its antitumorigenic function in breast cancer.
Assuntos
Neoplasias da Mama , Proteína Supressora de Tumor p53 , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/metabolismo , Fosforilação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Several reports have shown that thymoquinone (TQ) effectively attenuates angiogenesis in cancer cells, resulting in suppression of tumor growth. However, it is not yet clear whether TQ reduces hypoxia-inducible factor-1α (HIF-1α) expression in hypoxic cancer cells. Here, we found that TQ was a novel HIF-1α inhibitor through hypoxia response element (HRE)-luciferase assay-based large screening by using 502 natural compounds containing chemical library. TQ reduced HIF-1α protein levels in renal cancer cells; however, it did not affect the HIF-1α protein levels in the presence of proteasome inhibitor, MG132, indicating that the reduction effects of TQ on HIF-1α protein are mediated via the ubiquitination-proteasome dependent pathway. TQ boosted HIF-1α protein degradation, and the mechanism was revealed by inhibiting interaction between HSP90 and HIF-1α. TQ suppressed downstream genes of HIF-1α, indicating negative impact of TQ on HIF-1α transcriptional activities. In addition, TQ altered glucose, lactate, and ATP levels, leading to anaerobic metabolic disturbance. TQ induced apoptosis in hypoxic cancer cells as determined by crystal violet staining and flow cytometry for annexin V-stained cells. Taken together, we suggested that TQ is a potential anticancer agent targeting HIF-1α.
Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Apoptose/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/metabolismoRESUMO
In cancer, activation of X-box binding protein (XBP1) has a critical role in tumorigenesis and cancer progression. Transcriptional regulatory mechanism of XBP1 in cancer development has been well known, however, regulation of ubiquitination and degradation of XBP1 has not been elucidated yet. Here we show that Fbw7, a substrate recognition component of the SKP1-Cullin-F-box-type E3 ligase, interacts with XBP1 in a phosphorylation-dependent manner, and facilitates XBP1 ubiquitination and protein degradation. Moreover, Fbw7 inhibits oncogenic pathways including NF-κB, AP1, and Myc induced by XBP1. Interestingly, XBP1 negatively regulates transcription of Fbw7 via a feedback mechanism through NF-κB/E2F-1 axis signaling pathway, suggesting that overexpression of XBP1s may contribute to low level of Fbw7 expression in human cancers. Therefore, a negative feedback loop between Fbw7 and XBP1 contributes to the regulation of tumor development and can be an attractive target for novel therapy in cancers.
RESUMO
In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.
RESUMO
YAP/TAZ, a transcriptional co-activator of Hippo pathway, has emerged as a central player in vessel homeostasis such as sprouting angiogenesis and vascular barrier stabilization, during development. However, the role of YAP/TAZ in pathological angiogenesis remains unclear. Here, we demonstrated that YAP/TAZ is a critical mediator in leukocyte-endothelial adhesion induced by the vascular inflammatory cytokine TNF-α. YAP/TAZ was dephosphorylated, translocated from the cytosol to the nucleus, and activated by TNF-α in endothelial cells. A specific inhibitor of Rho GTPases suppressed the TNF-α-induced dephosphorylation of YAP. Knockdown of YAP/TAZ using siRNA significantly reduced the expression of the leukocyte adhesion molecule VCAM1 induced by TNF-α. The adhesion of monocytes to endothelial cells was also markedly reduced by YAP/TAZ silencing. However, knockdown of YAP/TAZ did not affect TNF-α-induced NF-κB signaling. Overall, these results suggest that YAP/TAZ plays critical roles in regulating TNF-α-induced endothelial cell adhesive properties without affecting the NF-κB pathway, and implicate YAP/TAZ as a potential therapeutic target for treating inflammatory vascular diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos/citologia , Fosfoproteínas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adesão Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Reduced therapeutic efficacy of sorafenib, a first-generation multikinase inhibitor, is often observed during the treatment of advanced hepatocellular carcinoma (HCC). Emodin is an active component of Chinese herbs, and is effective against leukemia, lung cancer, colon cancer, pancreatic cancer, and HCC; however, the sensitizing effect of emodin on sorafenib-based HCC therapy has not been evaluated. Here, we demonstrate that emodin significantly improved the anti-cancer effect of sorafenib in HCC cells, such as HepG2, Hep3B, Huh7, SK-HEP-1, and PLC/PRF5. Mechanistically, emodin inhibits sterol regulatory element-binding protein-2 (SREBP-2) transcriptional activity, which suppresses cholesterol biosynthesis and oncogenic protein kinase B (AKT) signaling. Additionally, attenuated cholesterol synthesis and oncogenic AKT signaling inactivated signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. Furthermore, emodin synergistically increased cell cycle arrest in the G1 phase and apoptotic cells in the presence of sorafenib. Animal models xenografted with HepG2 or SK-HEP-1 cells also showed that the combination of emodin and sorafenib was sufficient to inhibit tumor growth. Overall, these results suggested that the combination of emodin and sorafenib may offer a potential therapy for patients with advanced HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Colesterol/metabolismo , Emodina/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Sorafenibe/administração & dosagem , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Emodina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As life expectancy increases, elderly populations tend to spend an increasing number of years in poor health, with chronic age-related diseases and disability. Therefore, the development of therapeutic strategies to treat or prevent multiple pathophysiological conditions in the elderly may improve health-adjusted life expectancy and alleviate the potential economic and social burdens arising from age-related diseases. Bioactive natural products might represent promising new drug candidates for the treatment of many chronic age-related diseases, including cancer, Alzheimer's disease, cardiovascular disease, obesity, and liver disease. Here, we discuss a therapeutic option using saikosaponins, which are triterpene saponins isolated from Bupleurum, against a variety of age-related diseases. Understanding the underlying mechanisms of natural products like saikosaponins in the treatment of age-related diseases may help in the development of diverse natural product-derived compounds that may be effective against a number of chronic health problems.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Ácido Oleanólico/análogos & derivados , Saponinas/uso terapêutico , Apoptose/efeitos dos fármacos , Doenças Cardiovasculares/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Humanos , Hepatopatias/tratamento farmacológico , Hepatopatias/patologia , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Obesidade/patologia , Obesidade/prevenção & controle , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Saponinas/química , Saponinas/farmacologiaRESUMO
Saikosaponin a (SSa), a bioactive phytochemical from Bupleurum, triggers sequential caspase-2 and caspase-8 activation, and thereby induces caspase-mediated apoptosis in human colon carcinoma (HCC) cells. However, the upstream mechanism of caspase-2 activation remains unknown. Therefore, we investigated the signaling mechanisms underlying SSa-induced caspase activation and apoptosis in HCC cells. SSa treatment triggered marked antitumor effects, especially in HCC cells, in a cell culture model and a mouse xenograft model. SSa also induced the activation of several endoplasmic reticulum (ER) stress signals. Specifically, caspase-4, a critical regulator of ER stress-induced apoptosis, was activated significantly after SSa treatment. Mechanistically, selective inhibition of caspase-4 suppressed SSa-induced apoptosis, colony inhibition, and the activation of caspase-3, -8, and -2, but not vice versa. Consistent with the important role of caspase-2 in the DNA damage response, SSa induced DNA damage, as evidenced by a cytokinesis-block micronucleus assay, single-cell gel electrophoresis, and an increase in the levels of γ-H2AX, a DNA damage marker. Moreover, inhibition of caspase-4 activation inhibited SSa-induced histone H2AX phosphorylation. Taken together, these results suggest that caspase-4 is an upstream regulator of SSa-induced DNA damage and caspase activation in HCC cells. Given that SSa-induced apoptosis appeared to be specific to certain cell types including HCC cells, SSa may be a promising cancer therapy agent in certain types of cancer.
RESUMO
Phosphorylation, the addition of a phosphate group to a molecule, is an effective way of regulating the biological properties of that molecule. Protein phosphorylation is a post-translational modification of proteins and affects cellular signaling transduction. Protein kinases induce phosphorylation by catalyzing the transfer of phosphate groups to serine, threonine, and tyrosine residues on protein substrates. Consistent with their roles in cancer, protein kinases have emerged as one of the most clinically useful target molecules in pharmacological cancer therapy. Intrinsic or acquired resistance of cancers against anti-cancer therapeutics, such as ionizing radiation, is a major obstacle for the effective treatment of many cancers. In this review, we describe key aspects of various kinases acting on proteins. We also discuss the roles of protein kinases in the pathophysiology and treatment of cancer. Because protein kinases correlate with radiation resistance in various types of cancer, we focus on several kinases responsible for radiation resistance and/or sensitivity and their therapeutic implications. Finally, we suggest some ongoing radiation-sensitization strategies using genetic loss and/or kinase inhibitors that can counteract radiation resistance-related protein kinases.
Assuntos
Neoplasias/radioterapia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/efeitos da radiação , Radiossensibilizantes/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Radiação IonizanteRESUMO
Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated. In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells. Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR). It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis. To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed. Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A. Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment. In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway.
Assuntos
Benzaldeídos/farmacologia , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Melanoma/genética , Melanoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Linhagem Celular Tumoral , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação TranscricionalRESUMO
Salmonella have been experimentally used as anti-cancer agents, because they show selective growth in tumours. In this study, we genetically modified attenuated Salmonella typhimurium to express and secrete interferon-gamma (IFN-γ) as a tumouricidal agent to enhance the therapeutic efficacy of Salmonella. IFN-γ was fused to the N-terminal region (residues 1-160) of SipB (SipB160) for secretion from bacterial cells. Attenuated S. typhimurium expressing recombinant IFN-γ (S. typhimurium (IFN-γ)) invaded the melanoma cells and induced cytotoxicity. Subcutaneous administration of S. typhimurium (IFN-γ) also efficiently inhibited tumour growth and prolonged the survival of C57BL/6 mice bearing B16F10 melanoma compared with administration of phosphate-buffered saline (PBS), unmodified S. typhimurium or S. typhimurium expressing empty vector (S. typhimurium [Vec]) in a natural killer (NK) cell-dependent manner. Moreover, genetically modified Salmonella, including S. typhimurium (IFN-γ), showed little toxicity to normal tissues with no observable adverse effects. However, S. typhimurium (IFN-γ)-mediated tumour suppression was attributed to direct killing of tumour cells rather than to stable anti-tumour immunity. Collectively, these results suggest that tumour-targeted therapy using S. typhimurium (IFN-γ) has potential for melanoma treatment.
Assuntos
Imunoterapia/métodos , Interferon gama/biossíntese , Melanoma Experimental/terapia , Organismos Geneticamente Modificados/metabolismo , Salmonella typhimurium/metabolismo , Neoplasias Cutâneas/terapia , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Salmonella typhimurium/patogenicidade , Células Tumorais CultivadasRESUMO
Fascaplysin, a natural product isolated from marine sponges, is a potential candidate for the development of anti-cancer drugs. However, the mechanism underlying its therapeutic effect of strengthening anti-cancer efficacy of other drugs is poorly understood. Here, we found that fascaplysin increases phosphorylation of protein kinase B (PKB), also known as AKT, and adenosine monophosphate-activated protein kinase (AMPK), which are considered therapeutic targets for cancer treatment due to their anti-apoptotic or pro-survival functions in cancer. A cell viability assay revealed that pharmacological suppression of AKT using LY294002 enhanced the anti-cancer effect of fascaplysin in various cancer cells. Similarly, fascaplysin was observed to have improved anti-cancer effects in combination with compound C, a selective AMPK inhibitor. Another challenge showed that fascaplysin increased the efficacy of methotrexate (MTX)-mediated cancer therapy by suppressing genes related to folate and purine metabolism. Overall, these results suggest that fascaplysin may be useful for improving the anti-cancer efficacy of targeted anti-cancer drugs, such as inhibitors of phosphoinositide 3-kinase AKT signaling, and chemotherapeutic agents, such as MTX.
Assuntos
Adenilato Quinase/antagonistas & inibidores , Antineoplásicos/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Adenilato Quinase/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metotrexato/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
USP7 is a deubiquitinating enzyme that involves the ubiquitin proteasome system (UPS) to maintain regulation of protein synthesis and degradation. The well-known substrate of USP7 is the Mdm2-p53 complex. In fact, several studies have reported that functional inhibition of USP7 induces cancer cell apoptosis through activation of p53. However, the contribution of oxidative or endoplasmic reticulum (ER) stress, which is commonly induced by inhibition of the UPS for USP7 inhibitor-mediated apoptosis in cancer cells, has not been investigated. In contrast to previous reports, we show that p53 is not critical during USP7 inhibitor-induced apoptosis in several cancer cells. Inhibition of deubiquitinating enzyme activities by USP7 inhibitors causes ER stress by accumulating polyubiquitinated proteins in cancer cells. Furthermore, we demonstrate that USP7 inhibitors increase intracellular reactive oxygen species and mainly cause cancer cell apoptosis. Taken together, our results reveal that oxidative and ER stress, rather than the Mdm2-p53 axis, mainly contributes to USP7 inhibitor-mediated apoptosis in cancer cells.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Experimentais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina , Ubiquitinação/efeitos dos fármacosRESUMO
Recent studies have shown anticancer activity of apigenin by suppressing glucose transporter 1 (GLUT1) expression in cultured cancer cells; however, it is not clear whether apigenin can suppress glucose metabolism in lung cancer cells or sensitize them to inhibition of glutamine utilization-mediated apoptosis through metabolic and oxidative stress. We show that apigenin significantly decreases GLUT1 expression in mice. Furthermore, we demonstrate that apigenin induces growth retardation and apoptosis through metabolic and oxidative stress caused by suppression of glucose utilization in lung cancer cells. The underlying mechanisms were defined that the anticancer effects of apigenin were reversed by ectopic GLUT1 overexpression and galactose supplementation, through activation of pentose phosphate pathway-mediated NADPH generation. Importantly, we showed that severe metabolic stress using a glutaminase inhibitor, compound 968, was involved in the mechanism of sensitization by apigenin. Taken together, the combination of apigenin with inhibitors of glutamine metabolism may provide a promising therapeutic strategy for cancer treatment.
Assuntos
Apoptose/efeitos dos fármacos , Transportador de Glucose Tipo 1/biossíntese , Neoplasias Pulmonares/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Animais , Apigenina/administração & dosagem , Benzofenantridinas/administração & dosagem , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Glutamina/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , NADP/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ionizing radiation (IR), such as X-rays and gamma (γ)-rays, mediates various forms of cancer cell death such as apoptosis, necrosis, autophagy, mitotic catastrophe, and senescence. Among them, apoptosis and mitotic catastrophe are the main mechanisms of IR action. DNA damage and genomic instability contribute to IR-induced cancer cell death. Although IR therapy may be curative in a number of cancer types, the resistance of cancer cells to radiation remains a major therapeutic problem. In this review, we describe the morphological and molecular aspects of various IR-induced types of cell death. We also discuss cytogenetic variations representative of IR-induced DNA damage and genomic instability. Most importantly, we focus on several pathways and their associated marker proteins responsible for cancer resistance and its therapeutic implications in terms of cancer cell death of various types and characteristics. Finally, we propose radiation-sensitization strategies, such as the modification of fractionation, inflammation, and hypoxia and the combined treatment, that can counteract the resistance of tumors to IR.
Assuntos
Aberrações Cromossômicas , Terapia Combinada/métodos , Raios gama/uso terapêutico , Neoplasias/terapia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/uso terapêutico , Apoptose/genética , Apoptose/efeitos da radiação , Autofagia/genética , Autofagia/efeitos da radiação , Análise Citogenética , Dano ao DNA/efeitos da radiação , Instabilidade Genômica , Humanos , Mitose/efeitos da radiação , Necrose/genética , Necrose/patologia , Neoplasias/genética , Neoplasias/patologia , Terapia por Raios XRESUMO
The unique proline isomerase Pin1 is pivotal for protecting against age-dependent neurodegeneration in Alzheimer's disease (AD), with its inhibition providing a molecular link between tangle and plaque pathologies. Pin1 is oxidatively modified in human AD brains, but little is known about its regulatory mechanisms and pathological significance of such Pin1 modification. In this paper, our determination of crystal structures of oxidized Pin1 reveals a series of Pin1 oxidative modifications on Cys113 in a sequential fashion. Cys113 oxidization is further confirmed by generating antibodies specifically recognizing oxidized Cys113 of Pin1. Furthermore, Pin1 oxidation on Cys113 inactivates its catalytic activity in vitro, and Ala point substitution of Cys113 inactivates the ability of Pin1 to isomerize tau as well as to promote protein turnover of tau and APP. Moreover, redox regulation affects Pin1 subcellular localization and Pin1-mediated neuronal survival in response to hypoxia treatment. Importantly, Cys113-oxidized Pin1 is significantly increased in human AD brain comparing to age-matched controls. These results not only identify a novel Pin1 oxidation site to be the critical catalytic residue Cys113, but also provide a novel oxidative regulation mechanism for inhibiting Pin1 activity in AD. These results suggest that preventing Pin1 oxidization might help to reduce the risk of AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Anticorpos , Domínio Catalítico , Linhagem Celular Tumoral , Hipocampo/metabolismo , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Oxirredução , Peptidilprolil Isomerase/imunologia , Proteínas tau/metabolismoRESUMO
ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-L-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Ácidos Undecilênicos/farmacologia , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspase 6/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers, and is closely associated with poor prognosis and chemo- or radiotherapeutic resistance. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer.
Assuntos
Ciclo Celular/fisiologia , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Bulbo Olfatório/citologia , Bulbo Olfatório/enzimologia , Bulbo Olfatório/metabolismo , Proteína Oncogênica v-akt/química , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The medicinal plant Ulmus davidiana var. japonica has significant potential as a cancer chemoprevention agent. Catechin-7-O-xyloside (C7Ox) was purified from ultrafine U. davidiana var. japonica ethanol extract. In the present study, we investigated the apoptotic effect of C7Ox in the non-small cell lung cancer (NSCLC) cell line H1299. C7Ox treatment induced cell death and decreased plasma membrane integrity, an event typical of apoptosis. C7Ox-induced apoptosis was associated with the proteolytic activation of caspase-6, cleavage of poly(ADP-ribose) polymerase (PARP) and loss of mitochondrial membrane potential. C7Ox also induced the endoplasmic reticulum (ER) stress-regulated pro-apoptotic transcription factor CHOP. The suppression of CHOP expression significantly decreased C7Ox-induced cell death, LDH leakage and caspase-6 activation. Antitumor effects, evaluated based on protracted tumor regression, were observed when nude-mice bearing H1299 xenografts were treated with C7Ox. C7Ox-induced tumor regression was accompanied by enhanced expression of CHOP mRNA. Our data suggest that C7Ox can trigger mitochondrial-mediated apoptosis, and that ER stress is critical for C7Ox-induced apoptosis in H1299 NSCLC cells.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspase 6/biossíntese , Catequina/análogos & derivados , Catequina/farmacologia , Estresse do Retículo Endoplasmático , Neoplasias Pulmonares/enzimologia , Mitocôndrias/efeitos dos fármacos , Xilose/análogos & derivados , Animais , Linhagem Celular Tumoral , Humanos , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Fator de Transcrição CHOP/biossíntese , Fator de Transcrição CHOP/genética , Xilose/farmacologiaRESUMO
The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis.