RESUMO
Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are becoming increasingly prevalent worldwide. Despite the different etiologies, their spectra and histological feature are similar, from simple steatosis to more advanced stages such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Studies including peroxiredoxin knockout models revealed that oxidative stress is crucial in these diseases, which present as consequences of redox imbalance. Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes that are major targets of reactive oxygen species (ROS) because of an oxidation-susceptible nucleophilic cysteine in their active site. Herein, we review the oxidative inactivation of two tumor suppressor PTPs, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and T-cell protein tyrosine phosphatase (TCPTP), and their contribution to the pathogenicity of ALD and NAFLD, respectively. This review might provide a better understanding of the pathogenic mechanisms of these diseases and help develop new therapeutic strategies to treat fatty liver disease.
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Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known as a tumor suppressor gene that is frequently mutated in numerous human cancers and inherited syndromes. PTEN functions as a negative regulator of PI3K/Akt signaling pathway by dephosphorylating phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) to phosphatidylinositol (4, 5)-bisphosphate (PIP2), which leads to the inhibition of cell growth, proliferation, cell survival, and protein synthesis. PTEN contains a cysteine residue in the active site that can be oxidized by peroxides, forming an intramolecular disulfide bond between Cys124 and Cys71. Redox regulation of PTEN by reactive oxygen species (ROS) plays a crucial role in cellular signaling. Peroxiredoxins (Prxs) are a superfamily of peroxidase that catalyzes reduction of peroxides and maintains redox homeostasis. Mammalian Prxs have 6 isoforms (I-VI) and can scavenge cellular peroxides. It has been demonstrated that Prx I can preserve and promote the tumor-suppressive function of PTEN by preventing oxidation of PTEN under benign oxidative stress via direct interaction. Also, Prx II-deficient cells increased PTEN oxidation and insulin sensitivity. Furthermore, Prx III has been shown to protect PTEN from oxidation induced by 15s-HpETE and 12s-HpETE, these are potent inflammatory and pro-oxidant mediators. Understanding the tight connection between PTEN and Prxs is important for providing novel therapies. Herein, we summarized recent studies focusing on the relationship of Prxs and the redox regulation of PTEN.
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Hepatic steatosis and subsequent fatty liver disease are developed in response to alcohol consumption. Reactive oxygen species (ROS) are thought to play an important role in the alcoholic fatty liver disease (AFLD). However, the molecular targets of ROS and the underlying cellular mechanisms are unknown. Here, we investigate roles of peroxiredoxin III and redox regulation of phosphatase and tension homolog deleted on chromosome 10 (PTEN) in the alcoholic fatty liver. Alcohol-induced mitochondrial oxidative stress was found to contribute to reversible oxidation of PTEN, which results in Akt and MAPK hyperactivation with elevated levels of the lipogenesis regulators SREBP1c and PPARγ. Moreover, mitochondrial peroxiredoxin III was found to have antagonistic effects on lipogenesis via the redox regulation of PTEN by removing ROS, upon alcohol exposure. This study demonstrated that redox regulation of PTEN and peroxiredoxin III play crucial roles in the development of AFLD.
Assuntos
Fígado Gorduroso Alcoólico , Fígado Gorduroso Alcoólico/genética , Fígado Gorduroso Alcoólico/metabolismo , Humanos , Lipogênese , Fígado/metabolismo , Oxirredução , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Peroxirredoxina III/metabolismoRESUMO
BACKGROUND: Transforming growth factor-ß (TGF-ß) signaling is a double-edged sword in cancer development and progression. TGF-ß signaling plays a tumor suppressive role during the early stages of tumor development but promotes tumor progression in later stages. We have previously identified various mutations of TGF-ß receptor II (TßRII) in human oral squamous cell carcinoma (OSCC) samples. In the present study we analyzed I227T/N236D mutation of TßRII, which was detected in the metastatic lymph node of an OSCC patient. METHODS: The effect of I227T/N236D TßRII mutation on transcriptional activities was measured using DR26 cells, which lack functional TßRII. HSC2 human OSCC cells stably expressing wild-type and I227T/N236D mutant TßRII were generated and used to examine the effect of I227T/N236D TßRII mutation on xenograft tumor growth, in vitro cell proliferation, apoptosis, migration, and invasion. RESULTS: The I227T/N236D mutation of TßRII upregulated TGF-ß signaling and promoted xenograft tumor growth when compared with the wild-type, without affecting the in vitro proliferative capacities. To delineate the differences in proliferative capacities in vivo and in vitro, the apoptotic and survival signals were analyzed following curcumin treatment. Concomitant with apoptotic induction, epidermal growth factor receptor (EGFR) activation was observed upon curcumin treatment, which was further activated in I227T/N236D mutant transfectant cells when compared with wild-type cells. Enhanced EGFR activation correlated with cell survival and apoptotic resistance. Enhanced migratory and invasive capabilities of I227T/N236D mutant cells also depended on EGFR signaling. CONCLUSIONS: These results suggest that enhanced EGFR signaling via upregulated TGF-ß signaling shifted the balance toward survival and promoted cell migration and invasion in I227T/N236D mutant cells, elucidating the role of I227T/N236D mutation of TßRII in OSCC progression.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Bucais/patologia , Mutação , Fenótipo , Transdução de Sinais , TransfecçãoRESUMO
Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) is a potent tumor suppressor and often dysregulated in cancers. Cellular PTEN activity is restrained by the oxidation of active-site cysteine by reactive oxygen species (ROS). Recovery of its enzymatic activity predominantly depends on the availability of cellular thioredoxin (Trx) and peroxiredoxins (Prx), both are important players in cell signaling. Trx and Prx undergo redox-dependent conformational changes through the oxidation of cysteine residues at their active sites. Their dynamics are essential for protein functionality and regulation. In this review, we summarized the recent advances regarding the redox regulation of PTEN, with a specific focus on our current state-of-the-art understanding of the redox regulation of PTEN. We also proposed a tight association of the redox regulation of PTEN with Trx dimerization and Prx hyperoxidation, providing guidance for the identification of novel therapeutic targets.
Assuntos
Peroxirredoxinas , Tiorredoxinas , Cisteína , Oxirredução , PTEN Fosfo-Hidrolase , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Selenium is a vital trace element present as selenocysteine (Sec) in proteins that are, thus, known as selenoproteins. Humans have 25 selenoproteins, most of which are functionally characterized as oxidoreductases, where the Sec residue plays a catalytic role in redox regulation and antioxidant activity. Glutathione peroxidase plays a pivotal role in scavenging and inactivating hydrogen and lipid peroxides, whereas thioredoxin reductase reduces oxidized thioredoxins as well as non-disulfide substrates, such as lipid hydroperoxides and hydrogen peroxide. Selenoprotein R protects the cell against oxidative damage by reducing methionine-R-sulfoxide back to methionine. Selenoprotein O regulates redox homeostasis with catalytic activity of protein AMPylation. Moreover, endoplasmic reticulum (ER) membrane selenoproteins (SelI, K, N, S, and Sel15) are involved in ER membrane stress regulation. Selenoproteins containing the CXXU motif (SelH, M, T, V, and W) are putative oxidoreductases that participate in various cellular processes depending on redox regulation. Herein, we review the recent studies on the role of selenoproteins in redox regulation and their physiological functions in humans, as well as their role in various diseases.
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Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase that coordinates various cellular processes. Its activity is regulated by the reversible oxidation of an active-site cysteine residue by H2O2 and thioredoxin. However, the potential role of lipid peroxides in the redox regulation of PTEN remains obscure. To evaluate this, 15-hydroperoxy-eicosatetraenoic acid (15s-HpETE), a lipid peroxide, was employed to investigate its effect on PTEN using molecular and cellular-based assays. Exposure to 15s-HpETE resulted in the oxidation of recombinant PTEN. Reversible oxidation of PTEN was also observed in mouse embryonic fibroblast (MEF) cells treated with a 15s-HpETE and Lipofectamine mixture. The oxidative dimerization of thioredoxin was found simultaneously. In addition, the absence of peroxiredoxin III aggravated 15s-HpETE-induced PTEN oxidation in MEF cells. Our study provides novel insight into the mechanism linking lipid peroxidation to the etiology of tumorigenesis.
Assuntos
Leucotrienos/uso terapêutico , Peróxidos Lipídicos/uso terapêutico , PTEN Fosfo-Hidrolase/efeitos dos fármacos , Peroxirredoxina III/uso terapêutico , Animais , Humanos , Leucotrienos/farmacologia , Peróxidos Lipídicos/farmacologia , Camundongos , Oxirredução , Peroxirredoxina III/farmacologia , TransfecçãoRESUMO
Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.
Assuntos
Derivados de Benzeno/farmacologia , Carcinógenos/farmacologia , Fibroblastos/efeitos dos fármacos , PTEN Fosfo-Hidrolase/química , Tiorredoxina Redutase 1/metabolismo , Tiorredoxinas/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , NADP/metabolismo , Oxirredução , Estresse Oxidativo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxina Redutase 1/genética , Tiorredoxinas/genéticaRESUMO
Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert-butyl hydroperoxide (t-BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.
Assuntos
Peróxido de Hidrogênio/farmacologia , PTEN Fosfo-Hidrolase/química , terc-Butil Hidroperóxido/farmacologia , Células HeLa , Humanos , Oxirredução , PTEN Fosfo-Hidrolase/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Tiorredoxinas/metabolismoRESUMO
Tissue microenvironment adjusts biological properties of different cells by modulating signaling pathways and cell to cell interactions. This study showed that epithelial-mesenchymal transition (EMT)/ mesenchymal-epithelial transition (MET) can be modulated by altering culture conditions. HPV E6/E7-transfected immortalized oral keratinocytes (IHOK) cultured in different media displayed reversible EMT/MET accompanied by changes in cell phenotype, proliferation, gene expression at transcriptional, and translational level, and migratory and invasive activities. Cholera toxin, a major supplement to culture medium, was responsible for inducing the morphological and biological changes of IHOK. Cholera toxin per se induced EMT by triggering the secretion of interleukin 6 (IL-6) from IHOK. We found IL-6 to be a central molecule that modulates the reversibility of EMT based not only on the mRNA level but also on the level of secretion. Taken together, our results demonstrate that IL-6, a cytokine whose transcription is activated by alterations in culture conditions, is a key molecule for regulating reversible EMT/MET. This study will contribute to understand one way of cellular adjustment for surviving in unfamiliar conditions.
Assuntos
Toxina da Cólera/farmacologia , Meios de Cultura/química , Interleucina-6/genética , Interleucina-6/metabolismo , Queratinócitos/citologia , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Microambiente Celular , Transição Epitelial-Mesenquimal , Humanos , Queratinócitos/transplante , Células MCF-7 , Transplante de Neoplasias , Fenótipo , Regulação para Cima , Peixe-Zebra/embriologiaRESUMO
PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , PTEN Fosfo-Hidrolase/análise , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Oxirredução , PTEN Fosfo-Hidrolase/genética , Coelhos , Proteínas Supressoras de Tumor/genéticaRESUMO
We present a novel gain-of-function mutation of TGF-ß receptor II (TßRII) found in human oral squamous cell carcinoma (OSCC). Expression of E221V/N238I mutant TßRII enhanced TGF-ß signaling. Mutation of TßRII conferred cells higher migratory and invasive capabilities and MMP-2 activity. In mouse tumor model, mutant tumors exhibited poor differentiation and E-cadherin relocalization to the cytosol. Lipid-raft-dependent endocytosis of TßRII was attenuated in mutant TßRII, suggesting that enhancement of TGF-ß signaling by this mutation is due to delayed TßRII internalization. Taken together, our results show a novel gain-of-function TßRII mutation, which enhances TGF-ß signaling leading to more invasive phenotypic changes in human OSCC.
Assuntos
Carcinoma de Células Escamosas/fisiopatologia , Neoplasias Bucais/fisiopatologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Nus , Neoplasias Bucais/genética , Mutação , Reação em Cadeia da Polimerase , Transdução de SinaisRESUMO
KAI1/CD82, a tetraspanin membrane protein functions as a metastasis suppressor in many types of human cancers and has been shown to regulate cell adhesion properties. In the present study, we investigated the underlying mechanism of KAI1/CD82-mediated changes in cell adhesion to the extracellular matrix using human prostate cancer cells. We found that high KAI1/CD82 expression attenuated short-term cell adhesion to uncoated- or fibronectin-coated plates. Moreover, high KAI1/CD82 expression generated an extracellular environment unfavorable for cell adhesion as compared to low KAI1/CD82 expression, suggesting KAI1/CD82-dependent regulation of extracellular matrix (ECM) molecule(s) expression and/or secretion. Among ECM components examined, fibronectin exhibited decreased expression and secretion in high KAI1/CD82-expressing cells. Furthermore, high KAI1/CD82 expression interfered with the activation of ß (1) integrin at the cell surface while total ß (1) integrin levels remained unchanged, concomitant with reduced formation of focal adhesion complex and decreased bundling of actin filaments. Finally, high KAI1/CD82 expression significantly retarded cell motility in a scratch wound assay. Taken together, our results strongly suggest that KAI1/CD82 attenuates the activation of ß (1) integrin, and thereby down-regulates outside-in signaling of ß (1) integrin, leading to the reduction of focal adhesion formation and fibronectin expression/secretion, which subsequently interferes with cell adhesion properties and motility.
Assuntos
Fibronectinas/antagonistas & inibidores , Adesões Focais/metabolismo , Integrina beta1/metabolismo , Proteína Kangai-1 , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Western Blotting , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica , Inativação Gênica , Humanos , Integrina beta1/genética , Proteína Kangai-1/genética , Proteína Kangai-1/metabolismo , Lipídeos , Masculino , Microscopia Confocal , Plasmídeos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/secundário , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transfecção , Regulação para CimaRESUMO
The activity of matrix metalloproteinases (MMPs), which selectively degrades the extracellular matrix (ECM), is critical in angiogenesis. Conversely, changes in ECM composition/structure alter the expression and activity of MMPs in various cell types. In the present study, we examined whether changes in ECM composition affect MMPs expression/activity of endothelial cells and thereby alter the surrounding ECM structure. Among the ECM molecules examined, fibronectin (FN) and vitronectin (VN) increased the expression and activity of MMP-9 in human umbilical vein endothelial cells (HUVECs). Both α(5)ß(1) and α(v)ß(3) integrins were involved in FN-induced MMP-9 expression. Also, FN-induced MMP-9 expression was found to be mediated by AP-1 transcription factors, including c-Jun, JunB, and JunD. Inhibitors or siRNAs specific to AP-1 activating signal transducers, including FAK-Src, PI3K/Akt, ERK, and JNK, abolished both FN-induced AP-1 activation and MMP-9 expression. VN-induced AP-1 activation and MMP-9 expression were also mediated by these AP-1 activating signal transducers in addition to p38 MAPK. Moreover, treatment with FN or VN resulted in increased degradation of collagen on HUVEC culture plates. Taken together, our data suggest that both fibronectin and vitronectin induce MMP-9 expression via the AP-1-activating signaling pathways in endothelial cells, and thereby stimulate degradation of surrounding collagen, leading to alterations in ECM structure and potentially the promotion of angiogenesis.
Assuntos
Endotélio Vascular/metabolismo , Fibronectinas/farmacologia , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/metabolismo , Fator de Transcrição AP-1/metabolismo , Vitronectina/farmacologia , Células Cultivadas , Colágeno/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição AP-1/genética , Veias Umbilicais/citologiaRESUMO
A cancer/testis antigen, CAGE, is widely expressed in various cancer tissues and cancer cell lines but not in normal tissues except the testis. In the present study, ectopic expression of CAGE in fibroblast cells resulted in foci formation, suggesting its cell-transforming ability. Using stable HeLa transfectant clones with the tetracycline-inducible CAGE gene, we found that CAGE overexpression stimulated both anchorage-dependent and -independent cell growth in vitro and promoted tumor growth in a xenograft mouse model. Cell cycle analysis showed that CAGE augments the levels of cyclin D1 and E, thereby activating cyclin-associated cyclin-dependent kinases and subsequently accelerating the G(1) to S progression. Moreover, increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even in a growth arrested state, indicating a direct effect of CAGE on G(1) cyclin expression. CAGE-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced retinoblastoma phosphorylation and subsequent E2F-1 nuclear translocation. In contrast, small interfering RNA-mediated knockdown of CAGE suppressed the expression of G(1) cyclins, activation of AP-1 and E2F-1, and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclins D1 and E.
Assuntos
Proliferação de Células , Ciclina D1/metabolismo , Ciclina E/metabolismo , RNA Helicases DEAD-box/fisiologia , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Western Blotting , Transformação Celular Neoplásica , Imunoprecipitação da Cromatina , Ciclina D1/genética , Ciclina E/genética , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/genética , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Feminino , Fase G1 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Isoliquiritigenin, a natural flavonoid found in licorice, shallots, and bean sprouts, has been demonstrated to inhibit proliferation and to induce apoptosis in a variety of human cancer cells. We attempted to ascertain the underlying mechanism by which isoliquiritigenin induced cell cycle arrest and cytotoxicity in HeLa human cervical cancer cells. Isoliquiritigenin treatment arrested cells in both G2 and M phase. The cells arrested in interphase (G2) showed markers for DNA damage including the formation of gamma-H2AX foci and the phosphorylation of ATM and Chk2, whereas the cells arrested in M phase evidenced separate poles and mitotic metaphase-like spindles with partially unaligned chromosomes. The induction of DNA damage and blockade at the metaphase/anaphase transition implied that isoliquiritigenin might function as a topoisomerase II poison, which was further demonstrated via an in vitro topoisomerase II inhibition assay. These results show that isoliquiritigenin inhibits topoiosmerase II activity, and the resultant DNA damage and arrest in mitotic metaphase-like stage contributes to the antiproliferative effects of isoliquiritigenin.
Assuntos
Anáfase/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Chalconas/farmacologia , Dano ao DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Anáfase/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Dano ao DNA/fisiologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G2/fisiologia , Células HeLa , Histonas/metabolismo , Humanos , Metáfase/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/fisiologia , Quinase do Ponto de Checagem 2 , DNA/efeitos dos fármacos , Dicetopiperazinas , Células HeLa , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Development of sensitive methods to monitor and quantitatively assess the expression levels of endogenous genes and the association-interaction of proteins in living cells and whole organisms is a complex and challenging problem. In this chapter, we have described basic methods for investigating protein-protein interactions which include immunoprecipitation, GST pull-down assays, peptide bead pull-down assays, chemical crosslinking and photoaffinity labeling. These methods should provide important tools to dissect crosstalk between proteins and the direct implications of this crosstalk in signaling pathways and cancer biology.