RESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is pivotal in development, metabolic homeostasis, and immune responses. While recent research has highlighted AhR's significant role in modulating oxidative stress responses, its mechanistic relationship with ferroptosis-an iron-dependent, non-apoptotic cell death-remains to be fully elucidated. In our study, we discovered that AhR plays a crucial role in ferroptosis, in part by transcriptionally regulating the expression of the solute carrier family 7 member 11 (SLC7A11). Our findings indicate that both pharmacological inactivation and genetic ablation of AhR markedly enhance erastin-induced ferroptosis. This enhancement is achieved by suppressing SLC7A11, leading to increased lipid peroxidation. We also obtained evidence of post-translational modifications of SLC7A11 during ferroptosis. Additionally, we observed that indole 3-pyruvate (I3P), an endogenous ligand of AhR, protects cells from ferroptosis through an AhR-dependent mechanism. Based on these insights, we propose that AhR transcriptionally regulates the expression of SLC family genes, which in turn play a pivotal role in mediating ferroptosis. This underscores AhR's essential role in suppressing lipid oxidation and ensuring cell survival under oxidative stress.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Ferroptose , Receptores de Hidrocarboneto Arílico , Transdução de Sinais , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Humanos , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação da Expressão Gênica , Piperazinas/farmacologiaRESUMO
Ras proteins are small GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. Mutations or deregulated activities of Ras are frequently the driving force for oncogenic transformation and tumorigenesis. Posttranslational modifications play a crucial role in mediating the stability, activity, or subcellular localization/trafficking of numerous cellular regulators including Ras proteins. A series of recent studies reveal that Ras proteins are also regulated by sumoylation. All three Ras protein isoforms (HRas, KRas, and NRas) are modified by SUMO3. The conserved lysine42 appears to be the primary site for mediating sumoylation. Expression of KRasV12/R42 mutants compromised the activation of the Raf/MEK/ERK signaling axis, leading to a reduced rate of cell migration and invasion in vitro in multiple cell lines. Moreover, treatment of transformed pancreatic cells with a SUMO E2 inhibitor blocks cell migration in a concentration-dependent manner, which is associated with a reduced level of both KRas sumoylation and expression of mesenchymal cell markers. Furthermore, mouse xenograft experiments reveal that expression of a SUMO-resistant mutant appears to suppress tumor development in vivo. Combined, these studies indicate that sumoylation functions as an important mechanism in mediating the roles of Ras in cell proliferation, differentiation, and malignant transformation and that the SUMO-modification system of Ras oncoproteins can be explored as a new druggable target for various human malignancies.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transdução de Sinais/fisiologia , Sumoilação/fisiologia , Proteínas ras/metabolismo , Animais , HumanosRESUMO
Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anticancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediate its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our present studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial-mesenchymal transition and may provide new directions for Ras-related anticancer drug development.
Assuntos
Proteínas de Transporte/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas rap1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Células A549 , Proteínas de Transporte/química , Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Células HEK293 , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica/genéticaRESUMO
Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. In this report, we show that human keratinocyte cell line (HaCaT) treated with hydrogen peroxide displayed an increased activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes. Intriguingly, preincubation of the complete culture medium with hydrogen peroxide accelerated AhR activation and its downstream signaling. Subsequent mass spectrometric analysis reveals that the oxidant elicits the production of oxindole, a tryptophan catabolic product. We further demonstrate that 2-oxindole (a major form of oxindole) is capable of activating AhR, strongly suggesting that ROS may exert a significant impact on AhR signaling. Consistent with this, we also observe that hexavalent chromium [Cr(VI)], a heavy metal known to generate ROS in vivo, enhances AhR protein levels, as well as stimulates expression of CYP1A2 in an AhR-dependent manner. Significantly, we show that hydrogen peroxide and 2-oxindole induce expression of IDO1 and PD-L1, two immune checkpoint proteins. Given the role of IDO1 and PD-L1 in mediating T cell activity and/or differentiation, we postulate that ROS in the tumor microenvironment may play a crucial role in immune suppression via perturbing AhR signaling.
RESUMO
Ras proteins participate in multiple signal cascades, regulating crucial cellular processes, including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that Lys-42 plays an important role in mediating the modification. In the current study, we further investigated the role of Lys-42 in regulating cellular activities of K-Ras. Inducible expression of K-RasV12 led to the activation of downstream components, including c-RAF, MEK1, and extracellular signal-regulated kinases (ERKs), whereas expression of K-RasV12/R42 mutant compromised the activation of the RAF/MEK/ERK signaling axis. Expression of K-RasV12/R42 also led to reduced phosphorylation of several other protein kinases, including c-Jun N-terminal kinase (JNK), Chk2, and focal adhesion kinase (FAK). Significantly, K-RasV12/R42 expression inhibited cellular migration and invasion in vitro in multiple cell lines, including transformed pancreatic cells. Given that K-Ras plays a crucial role in mediating oncogenesis in the pancreas, we treated transformed pancreatic cells of both BxPC-3 and MiaPaCa-2 with 2-D08, a small ubiquitin-like modifier (SUMO) E2 inhibitor. Treatment with the compound inhibited cell migration in a concentration-dependent manner, which was correlated with a reduced level of K-Ras sumoylation. Moreover, 2-D08 suppressed expression of ZEB1 (a mesenchymal cell marker) with concomitant induction of ZO-1 (an epithelial cell marker). Combined, our studies strongly suggest that posttranslational modification(s), including sumoylation mediated by Lys-42, plays a crucial role in K-Ras activities in vivo.
Assuntos
Movimento Celular , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonas/farmacologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3 , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Sumoilação/efeitos dos fármacos , Sumoilação/genética , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Quantum dots (QDs) have shown great potential for biomedical use in a broad range including diagnostic agents. However, the regulatory mechanism of dermal toxicity is poorly understood. In this study, we investigated how QDs-induced apoptosis is regulated in human keratinocytes. We also examined the effect of carboxylic acid-coated QDs (QD 565 and QD 655) on reactive oxygen species (ROS) production and apoptosis-related cellular signalling. The viability of keratinocyte was inhibited by two types of QDs in a concentration-dependent manner. QDs induce ROS production and blockade of AKT phosphorylation. Moreover, the cleavage of AKT-dependent pro-apoptotic proteins such as poly (ADP-ribose) polymerase, caspases-3 and caspases-9 was significantly increased. We also found that a decrease in cellular ROS level by ROS scavenger, N-acetylcysteine (NAC), resulting in the abolishment of QDs-induced AKT de-phosphorylation and cellular apoptosis. Interestingly, QD 655 had a more cytotoxic effect including oxidative stress and AKT-dependent apoptosis than QD 565. In addition, QD 655 had the cytotoxic potential in the human skin equivalent model (HSEM). These data show that QD-induced intracellular ROS levels may be an important parameter in QD-induced apoptosis. These findings from this study indicate that intracellular ROS levels might determine the apoptotic potential of keratinocyte by QD via blockade of AKT phosphorylation.
Assuntos
Apoptose , Epiderme/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pontos Quânticos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Carboxílicos , Caspase 3/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular , Células Cultivadas , Humanos , Queratinócitos/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Pontos Quânticos/química , Transdução de SinaisRESUMO
A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on Ser-380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains, and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on Ser-380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, the PTEN interaction with Cdh1 was phosphatase-independent, and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Caderinas/metabolismo , Cromatina/metabolismo , Mitose/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Antígenos CD , Caderinas/genética , Cromatina/genética , Inibidores de Cisteína Proteinase/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/fisiologia , Células HeLa , Humanos , Leupeptinas/farmacologia , Mitose/efeitos dos fármacos , PTEN Fosfo-Hidrolase/genética , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologiaRESUMO
PTEN is a well known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pulldown analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated Ser-380, Thr-382, and Thr-383, but not Ser-385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only Ser-380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with the accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Cromatina/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , PTEN Fosfo-Hidrolase/química , Fosforilação , Serina/metabolismo , Quinase 1 Polo-LikeRESUMO
While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Proteínas Quinases Associadas a Fase S , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Humanos , Transdução de Sinais , Ciclo Celular/genética , Transição Epitelial-Mesenquimal/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de CélulasRESUMO
Chronic environmental exposure to toxic metal(loid)s significantly contributes to human cancer development and progression. It is estimated that approximately 90% of cancer deaths are a result of metastasis of malignant cells, which is initiated by epithelial-mesenchymal transition (EMT) during early carcinogenesis. EMT is regulated by many families of genes and microRNAs (miRNAs) that control signaling pathways for cell survival, death, and/or differentiation. Recent mechanistic studies have shown that toxic metal(loid)s alter the expression of miRNAs responsible for regulating the expression of genes involved in EMT. Altered miRNA expressions have the potential to be biomarkers for predicting survival and responses to treatment in cancers. Significantly, miRNAs can be developed as therapeutic targets for cancer patients in the clinic. In this mini review, we summarize key findings from recent studies that highlight chemical-miRNA-gene interactions leading to the perturbation of EMT after exposure to toxic metal(loid)s including arsenic, cadmium, nickel, and chromium.
RESUMO
OBJECTIVES: Besides its role in regulating phosphatidylinositol-3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division. MATERIALS AND METHODS: Immunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle. RESULTS: Polycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co-localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated-BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells. CONCLUSION: CBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.
Assuntos
Proliferação de Células/fisiologia , Mitose/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Ciclo Celular/fisiologia , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Fosforilação , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
Curcumin, a constituent of turmeric, has anti-inflammatory, anti-carcinogenic, and chemopreventive effects in several animal tumor models. The expression of P-glycoprotein (P-gp), encoded by the mdr gene, is often associated with multidrug resistance (MDR) to unrelated chemotherapeutic drugs in cancer cells. Here, we demonstrate that curcumin down-regulates P-gp expression in multidrug-resistant L1210/Adr cells. Transfection with a series of 5'-deleted constructs of the mdr1b gene promoter indicated that a proximal region between -205 and +42 of the sequence was responsible for the suppression of promoter activity by curcumin. This response might be associated with the inhibition of the phosphatidyinositol 3-kinase (PI3K)/Akt/nuclear factor-kappa B (NF-kappa B) signaling pathway by curcumin. Moreover, curcumin reversed the MDR of the L1210/Adr cells. Thus, curcumin can contribute to the reversal of the MDR phenotype, probably due to the suppression of P-gp expression via the inhibition of the PI3K/Akt/NF-kappa B signaling pathway.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Curcumina/farmacologia , Leucemia L1210/tratamento farmacológico , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Curcumina/uso terapêutico , Relação Dose-Resposta a Droga , Regulação para Baixo , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia L1210/enzimologia , Leucemia L1210/metabolismo , Camundongos , NF-kappa B/antagonistas & inibidores , Células NIH 3T3 , Inibidores de Fosfoinositídeo-3 Quinase , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transfecção , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Tamoxifen (TAM) is a synthetic non-steroidal anti-estrogen compound that is widely used as an effective chemotherapeutic agent for treatment and prevention of breast cancer. Unfortunately, prolonged treatment with TAM causes TAM-responsive tumors to become TAM resistant through an as-yet-unknown mechanism. To develop novel anti-breast cancer agents that are therapeutically superior to TAM, we must first fully understand the biological effects of TAM. In this study, we found that TAM treatment of MDA-MB-361 breast cancer cells activated p21Waf1/Cip1 gene transcription independently of p53. Furthermore, TAM-induced p21Waf1/Cip1 promoter activity was enhanced by transient expression of the gene encoding Early Growth Response-1 (Egr-1) protein, a transcription factor that plays an important role in cell growth and differentiation. The TAM-induced p21Waf1/Cip1 promoter activity was blocked by the expression of small interfering RNA (siRNA) targeted to Egr-1 mRNA. In addition, induction of Egr-1 expression by TAM occurred at the transcriptional level via Ets-domain transcription factor Elk-1 through the JNK and p38 mitogen-activated protein (MAP) kinase pathways. Inhibition of the JNK and p38 MAP kinase signals inhibited Egr-1-mediated p21Waf1/Cip1 promoter activity. We conclude that TAM stimulation of p21Waf1/Cip1 gene transcription in MDA-MB-361 cells depends largely on Elk-1-mediated Egr-1 expression induced by activation of the JNK and p38 MAP kinase pathways.
Assuntos
Neoplasias da Mama/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Tamoxifeno/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
RAS proteins are GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, differentiation, and autophagy. Mutations or deregulated activities of RAS are frequently the driving force for oncogenic transformation and tumorigenesis. Given the important roles of the small ubiquitin-related modifier (SUMO) pathway in controlling the stability, activity, or subcellular localization of key cellular regulators, we investigated here whether RAS proteins are posttranslationally modified (i.e. SUMOylated) by the SUMO pathway. We observed that all three RAS protein isoforms (HRAS, KRAS, and NRAS) were modified by the SUMO3 protein. SUMOylation of KRAS protein, either endogenous or ectopically expressed, was observed in multiple cell lines. The SUMO3 modification of KRAS proteins could be removed by SUMO1/sentrin-specific peptidase 1 (SENP1) and SENP2, but not by SENP6, indicating that RAS SUMOylation is a reversible process. A conserved residue in RAS, Lys-42, was a site that mediates SUMOylation. Results from biochemical and molecular studies indicated that the SUMO-E3 ligase PIASγ specifically interacts with RAS and promotes its SUMOylation. Moreover, SUMOylation of RAS appeared to be associated with its activation. In summary, our study reveals a new posttranslational modification for RAS proteins. Since we found that HRAS, KRAS, and NRAS can all be SUMOylated, we propose that SUMOylation might represent a mechanism by which RAS activities are controlled.
RESUMO
Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3beta pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3beta and increased GSK-3beta kinase activity. A voltage-sensitive Ca(2+) channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca(2+) concentration prevented CZP-induced dephosphorylation of Akt and GSK-3beta, suggesting that Ca(2+)/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3beta, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca(2+) concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3beta pathway by counteracting Ca(2+)/CaM in PTEN-negative U-87MG glioblastoma cells.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Clozapina/farmacologia , Glioblastoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Antipsicóticos/farmacologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Deleção de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Proteínas Nucleares/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Multidrug resistance (MDR) to unrelated chemotherapeutic drugs can be mediated by overexpression of P-glycoprotein (P-gp), the mdr gene product. Trifluoperazine (TFP), a phenothiazine derivative antipsychotics, is known to reverse MDR of tumor cell lines by blocking P-gp efflux function. In the present study, we evaluated the effect of TFP on the expression of P-gp in multidrug-resistant L1210/Adr mouse leukemic cell lines, which are characterized by overexpession of P-gp. We found that TFP induced the downregulation of P-gp protein and mdr1b mRNA in a dose- and time-dependent manner in L1210/Adr cells. TFP reduction of mdr1b mRNA was paralleled by transcriptional suppression of the mdr1b promoter. Moreover, TFP restored the adriamycin-induced apoptosis in L1210/Adr cells. These results suggest that TFP may have utility as an adjuvant in the therapy of leukemia for the reversal of P-gp-dependent MDR as well as for the management of psychological symptoms in the cancer patients.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antipsicóticos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia/metabolismo , Trifluoperazina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia/genética , Camundongos , NF-kappa B/metabolismo , Células NIH 3T3 , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we have asked whether WWP2 plays a role in controlling cell cycle progression. Here we report that WWP2 is necessary for normal cell cycle progression as its silencing significantly reduces the cell proliferation rate. We have identified that an isoform of WWP2 (WWP2-V4) is highly expressed in the M phase of the cell cycle. Silencing of WWP2 accelerates the turnover of cyclin E, which is accompanied by increased levels of phospho-histone H3 (p-H3) and cyclin B. Moreover, silencing of WWP2 results in compromised phosphorylation of Akt(S473), a residue whose phosphorylation is tightly associated with the activation of the kinase. Combined, these results strongly suggest that WWP2 is an important component in regulating the Akt signaling cascade, as well as cell cycle progression.
RESUMO
A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN's role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3ß are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN's association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as γH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.