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1.
Int J Mol Med ; 37(3): 703-15, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26782291

RESUMO

Acute lung injury (ALI) as a serious diseases with high mortality and is considered a threat to human health and life. A number of studies have focused on the treatment and prevention of lung injury. However, the molecular mechanisms responsible for the development of lung injury are not yet fully understood, and this has impeded the development of effective drugs and treatment strategies. Hence, in the present study, mice with lipopolysaccharide (LPS)­induced ALI were used as a model to investigate the crosstalk between the CX3CL1-CX3CR1 axis and the nuclear factor (NF)-κB signaling pathway in the process of lung injury. CX3CL1-knockout (CX3CL1-KO or CX3CL1-/-) mice were used to examine the role of the CX3CL1-CX3CR1 axis in LPS-induced lung injury. We used baicalin, a natural product, to investigate its anti-inflammatory effects and its protective effects against lung injury. Western blot analysis, reverse transcription-quantitavie PCR (RT-qPCR), immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and the analysis of biochemical indicators were used to determine the key signaling pathway involved in the development of lung injury. The results indicated that, on the one hand, baicalin exerted potent anti-inflammatory effects by inhibiting the activation of the CX3CL1-CX3CR1 axis and NF-κB, thus preventing the the crosstalk between the CX3CL1­CX3CR1 axis and NF-κB pathway. In addition, the phosphorylation of AKT, which was significantly induced by LPS-induced ALI through the CX3CL1-CX3CR1 axis, was inhibited by treatment with baicalin. On the other hand, we further investigated the role of the CX3CL1-CX3CR1 axis in lung injury. We determined the diffrences in the expression levels of CX3CR1 between wild-type (WT) and CX3CL1-/- mice in order to establish its association with lung injury. Our results indicated that CX3CL1 may be the central and major indicator in the process of lung injury, which mediates the CX3CR1 receptor to activate AKT and further promote NF-κB activation. These findings demonstrate that the crosstalk between the CX3CL1-CX3CR1 axis and NF-κB signaling pathway plays a direct role in LPS-induced lung injury. The inhibition of the activation of the CX3CL1-CX3CR1 axis may thus suppress the development of ALI. In addition, baicalin inhibited the crosstalk between the CX3CL1-CX3CR1 axis and NF-κB pathway in mice with LPS-induced ALI. Thus, treatment with baicalin may be a potential therapeutic strategy for ALI.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Quimiocina CX3CL1/metabolismo , Flavonoides/uso terapêutico , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Receptores de Quimiocinas/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos
2.
Oncotarget ; 6(9): 7011-22, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25749521

RESUMO

TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Antineoplásicos/química , Ensaio Cometa , Dano ao DNA , Células HeLa , Humanos , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Radiação Ionizante , Serina/química , Tanquirases/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética
3.
Int J Biol Sci ; 9(4): 425-34, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23678292

RESUMO

The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnormal DNA damage response, including decreased IR-induced phosphorylation of H2AX, Chk1, Chk2 and Kap-1 as well as a prolonged G2-M arrest and aberrant mitotic progression. Notably, PIG3 knockdown resulted in a striking depression of cellular DNA-PKcs protein level, and was accompanied by a downregulation of ATM. Re-expression of PIG3 effectively rescued the depression of DNA-PKcs in PIG3-depleted cells. This negative regulation of DNA-PKcs by depleting PIG3 seemed to take place at the translational level but not at the levels of transcription or protein degradation. However, a compensatory feedback of increased mRNA expression of DNA-PKcs was formed in PIG3-depleted cells after a few passages or cell cycles of subculture, which led the recovery of the DNA-PKcs protein level and the consequent recovered efficiency of the DNA damage response. These results provide a new insight into the mechanism of PIG3's functioning in DNA damage signaling and the regulation network of cellular DNA-PKcs expression homeostasis.


Assuntos
Dano ao DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Citometria de Fluxo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas/genética , Radiação Ionizante
4.
Toxicol Appl Pharmacol ; 252(3): 307-17, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21419150

RESUMO

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3σ and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe.


Assuntos
Benzaldeídos/farmacologia , Neoplasias Hepáticas/metabolismo , Proteômica/métodos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Citometria de Fluxo , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Mitose/efeitos dos fármacos
5.
Radiat Oncol ; 5: 70, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20704701

RESUMO

BACKGROUND: Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. METHODS: DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of gammaH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells. RESULTS: Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of gammaH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy. CONCLUSION: The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential.


Assuntos
Proteína Quinase Ativada por DNA/antagonistas & inibidores , Neoplasias/genética , Neoplasias/terapia , Tolerância a Radiação/genética , Anticorpos de Cadeia Única/uso terapêutico , Animais , Especificidade de Anticorpos , Proliferação de Células , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Terapia Genética/métodos , Células HeLa , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Res ; 70(9): 3657-66, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20406977

RESUMO

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is well known as a critical component involving the nonhomologous end joining pathway of DNA double-strand breaks repair. Here, we showed another important role of DNA-PKcs in stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. Inactivation of DNA-PKcs by small interfering RNA or specific inhibitor NU7026 resulted in an increased outcome of polyploidy after 2-Gy or 4-Gy irradiation. Simultaneously, a high incidence of multinucleated cells and multipolar spindles was detected in DNA-PKcs-deficient cells. Time-lapse video microscopy revealed that depression of DNA-PKcs results in mitotic catastrophe associated with mitotic progression failure in response to DNA damage. Moreover, DNA-PKcs inhibition led to a prolonged G(2)-M arrest and increased the outcome of aberrant spindles and mitotic catastrophe in Ataxia-telangiectasia mutated kinase (ATM)-deficient AT5BIVA cells. We have also revealed the localizations of phosphorylated DNA-PKcs/T2609 at the centrosomes, kinetochores, and midbody during mitosis. We have found that the association of DNA-PKcs and checkpoint kinase 2 (Chk2) is driven by Ku70/80 heterodimer. Inactivation of DNA-PKcs strikingly attenuated the ionizing radiation-induced phosphorylation of Chk2/T68 in both ATM-efficient and ATM-deficient cells. Chk2/p-T68 was also shown to localize at the centrosomes and midbody. These results reveal an important role of DNA-PKcs on stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage and provide another prospect for understanding the mechanism coupling DNA repair and the regulation of mitotic progression.


Assuntos
Dano ao DNA/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Centrossomo/enzimologia , Quinase do Ponto de Checagem 2 , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Mitose/genética , Fosforilação , Poliploidia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo
7.
FASEB J ; 24(2): 495-503, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19825974

RESUMO

Cyclin B1, an important cell cycle regulator, was up-regulated in lymphocytes of human immunodeficiency virus (HIV)-infected patients. However, the mechanism of cyclin B1 up-regulation and the effects of the up-regulation on the host cells remain unclear. Here, we show that HIV-encoded Tat protein regulates cyclin B1 levels in two different ways: first, Tat stimulates the transcription of cyclin B1, which increases cyclin B1 levels and promotes the cells apoptosis; and second, Tat stimulates polyubiquitination-mediated degradation of cyclin B1 through binding to the N-terminal of cyclin B1 (aa 61-129) that is just downstream of the D box, which prevents excessive levels of cyclin B1 in the cells. These results suggest that Tat-regulating cyclin B1 affects the status of HIV: Tat stimulates cyclin B1 expression to slow down the host cell cycle progress and to promote the host cell apoptosis, which might facilitate HIV release; Tat stimulates cyclin B1 degradation to prevent overaccumulation of cyclin B1, which might facilitate HIV replication. Taken together, our results reveal for the first time how HIV-Tat regulates cyclin B1 and keeps its balance in the cells.


Assuntos
Apoptose/efeitos dos fármacos , Ciclina B1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologia , Linhagem Celular Tumoral , Ciclina B1/biossíntese , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/fisiopatologia , Humanos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
8.
Zhonghua Zhong Liu Za Zhi ; 31(10): 746-851, 2009 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-20021826

RESUMO

OBJECTIVE: To observe the anti-proliferation effect of bevacizumab and SN-38 (active metabolite of irinotecan), and investigate the possible mechanisms of these two agents. METHODS: Human colon cancer LoVo cells were cultured under hypoxic conditions. Inhibition of cell proliferation was evaluated by MTT assay. The drug modulation on HIF-1alpha, VEGF, ERK and AKT were assessed by the following assays. The mRNA expression of HIF-1alpha and VEGF were measured by RT-PCR. The protein expression of HIF-1alpha, ERK and AKT were evaluated by Western blot analysis, and VEGF by ELISA assay. RESULTS: Among different combination schedules, Bevacizumab given after SN-38 show most synergistic anti-proliferation effect. Under hypoxic conditions, the expression of HIF-1alpha and VEGF increased as time accumulated, Bevacizumab combined with SN-38 almost completely inhibited the expression of HIF-1alpha and VEGF. Moreover, the MAP kinase pathway was involved in the drug modulation of HIF-1alpha and VEGF. CONCLUSION: These findings suggest the anti-proliferation effect of bevacizumab and SN-38 was schedule-dependent, and the synergistic effect of Bevacizumab and SN-38 was related to drug modulation of the HIF-1alpha and MAP kinase pathway.


Assuntos
Anticorpos Monoclonais/farmacologia , Camptotecina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Anticorpos Monoclonais Humanizados , Antineoplásicos Fitogênicos/farmacologia , Bevacizumab , Camptotecina/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Irinotecano , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética
9.
Radiat Environ Biophys ; 48(2): 205-13, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19238419

RESUMO

The immediate-early response gene 5 (IER5) was previously shown, using microarray analysis, to be upregulated by ionizing radiation. Here we further characterized the dose- and time-dependency of radiation-induced expression of IER5 at doses from 0.5 to 15 Gy by quantitative real-time PCR analyses in HeLa cells and human lymphoblastoid AHH-1 cells. A radiation-induced increase in the IER5 mRNA level was evident 2 h after irradiation with 2 Gy in both cell lines. In AHH-1 cells the expression reached a peak at 4 h and then quickly returned to the control level, while in HeLa cells the expression only remained increased for a short period of time at around 2 h after irradiation before returning to the control. After high-dose irradiation (10 Gy), the induction of the IER5 expression was lower and delayed in AHH-1 cells as compared with 2-Gy irradiated cells. In HeLa cells, at this dose, two peaks of increased expression were observed 2 h and 12-24 h post-irradiation, respectively. RNA interference technology was employed to silence the IER5 gene in HeLa cells. siRNA-mediated suppression of IER5 resulted in an increased proliferation of HeLa cells. Cell growth and survival analyses demonstrated that suppression of IER5 significantly increased the radioresistance of HeLa cells to radiation doses of up to 6 Gy, but barely affected the sensitivity of cells at 8 Gy. Moreover, suppression of IER5 potentiated radiation-induced arrest at the G2-M transition and led to an increase in the fraction of S phase cells. Taken together, we propose that the early radiation-induced expression of IER5 affects the radiosensitivity via disturbing radiation-induced cell cycle checkpoints.


Assuntos
Ciclo Celular/efeitos da radiação , Raios gama , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces/biossíntese , Proteínas Nucleares/biossíntese , Linhagem Celular , Proliferação de Células , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Células HeLa , Humanos , Linfócitos/efeitos da radiação , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação , Fatores de Tempo , Transfecção
10.
Mol Cancer ; 7: 32, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18426604

RESUMO

BACKGROUND: C-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein. RESULTS: Firstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 beta on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3beta and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 beta, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells. CONCLUSION: A suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Proliferação de Células , Proteína Quinase Ativada por DNA/fisiologia , Regulação para Baixo , Instabilidade Genômica , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno , Transdução de Sinais
11.
Eur J Pharm Sci ; 33(1): 52-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17981442

RESUMO

Vanillin is a naturally occurring compound and food-flavoring agent with antioxidant and antimutagenic activities. In present study, we explored the radioprotective effect of a novel vanillin derivative VND3207 (4-hydroxy-3,5-dimethoxybenzaldehyde). VND3207 has a much higher potential in scavenging hydroxyl radical and superoxide radical than vanillin as indicated in the ESR spin-trapping measurement, and it can effectively protect plasmid DNA against 10-50 Gy gamma-ray induced breaks in vitro at the concentrations as low as 10-20 microM. Using human lymphoblastoid AHH-1 cells and human fibroblastoid HFS cells, we demonstrated that VND3207 at 5-40 microM concentrations significantly attenuated the inhibition of proliferation and occurrence of apoptosis produced by 1-8 Gy gamma-irradiation. In the cultured cells, VND3207 significantly decreased the initial production and residual level of DNA double-strand breaks (DSBs) induced by 2 or 8 Gy irradiation. Treatment of VND3207 enhanced the level of DNA-PKcs protein, a critical component of DNA DSB repair pathway in the cells with or without gamma-irradiation. Consistently, the phosphorylation of Akt protein, a mediator of survival signal, as well as its substrate GSK3beta was concurrently increased by VND3207. Our results suggest that VND3207 has radioprotection effect through its capabilities as a powerful antioxidant, in minimizing DNA damage, and activating survival signal Akt pathway, and it may be of value in the development of radioprotective compounds.


Assuntos
Benzaldeídos/farmacologia , Dano ao DNA/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Protetores contra Radiação/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Benzaldeídos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Ensaio Cometa/métodos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Citometria de Fluxo/métodos , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Linfócitos/efeitos da radiação , Masculino , Estrutura Molecular , Fosforilação , Protetores contra Radiação/química , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação
12.
Nan Fang Yi Ke Da Xue Xue Bao ; 27(9): 1303-6, 2007 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-17884763

RESUMO

OBJECTIVE: To study the effect of the small interfering RNA (siRNA)-mediated LKB1 gene silencing on the biological behavior of lung carcinoma cells. METHODS: RNA interference technique was used to silence LKB1 gene in lung carcinoma cells, and the expression of LKB1 protein were subsequently detected by Western blotting. The cell proliferation was then assessed by observing the growth curve and clone formation of the cells, and cell cycle and apoptosis were assayed by flow cytometry. RESULTS: LKB1 siRNA resulted in remarkable suppression of LKB1 expression in the lung carcinoma cells. LKB1 gene silencing resulted in accelerated cell proliferation, but cell apoptosis underwent no significant changes. CONCLUSION: Down-regulation of LKB1 gene expression can stimulate malignant biological behavior of lung carcinoma cells.


Assuntos
Inativação Gênica , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/genética
13.
Int J Mol Med ; 19(4): 607-15, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17334636

RESUMO

The sensitivity of cancer cells as well as normal cells in response to ionizing radiation (IR) is believed to be associated with the early inducible expression of specific genes. Using cDNA microarray technology, here we explored and compared the global transcriptional changes in human lymphoblastoid AHH-1 cells irradiated with 0.05-, 0.2-, 0.5-, 2.0- and 10-Gy doses of gamma-rays 4 h after exposure. A dose as low as 0.05 Gy was efficient in inducing a transcriptional response including the up-regulation of 25 genes, some of which are involved in signal transduction pathways, e.g. BMPR2, GPR124, MAPK8IP2 and AGGF1, and the down-regulation of 18 genes. Expression of some genes was altered only at a specific dose. Most importantly, we discovered a number of radiation-response genes, e.g. DNA repair gene XPC, tumor protein p53 inducible protein 3 gene (TP53I3), immediate early response 5 gene, whose transcriptional levels were increased or depressed by IR in a dose-dependent trend within the dose range 0.05-10 Gy. The dose-dependent induced expression of TP53I3 and XPC was confirmed by Northern blot analyses. Using quantitative real-time PCR, we further confirmed that XPC gene induction was dose dependent as well as time dependent, reaching a peak 4 h post-2 Gy and 10 h post-0.05 Gy. The maximum induced expression level of the XPC gene was higher after 2 Gy (3.2-fold) than 0.05 Gy (1.93-fold). The identification of these radiation-inducible genes, especially those exhibiting a dose-dependent response, not only expands our knowledge of the mechanisms underlying the diverse biological effects induced by IR, but provides candidates for developing novel biomarkers of radiation injury.


Assuntos
Reparo do DNA/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Expressão Gênica/efeitos da radiação , Linfoma/genética , Linhagem Celular Tumoral , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta à Radiação , Regulação para Baixo , Raios gama , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ativação Transcricional , Proteína Supressora de Tumor p53/genética
14.
Int J Oncol ; 29(5): 1167-72, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17016648

RESUMO

Vanillin, a naturally occurring food component, has been reported to have anti-mutagenic and anti-metastatic potentials, and to inhibit DNA-PKcs activity. However, vanillin itself exhibits very weak antiproliferative activity. We explored the effects of bromovanin (6-bromine-5-hydroxy-4-methoxybenzaldehyde), a novel vanillin derivative, on survival and cell-cycle progression of human Jurkat leukemia cells. Treatment with >10 microM bromovanin significantly elicited apoptosis and G2/M arrest in Jurkat cells in a dose- and time-dependent manner. Bromovanin-induced DNA double-strand breaks (DSB) were demonstrated by means of comet assay as well as detection of phosphorylated H2AX, a sensitive indicator of DNA DSBs. Immuno-hybridization analysis revealed that the cleavage of procaspase-3 and DNA-PKcs occurred concurrently with bromovanin-induced apoptosis. Furthermore, phosphorylated Akt protein (Ser473), which is catalyzed by DNA-PKcs, as well as phosphorylated GSK3beta (a substrate of activated Akt), markedly decreased in bromovanin-treated Jurkat cells, suggesting that bromovanin leads to inactivation of Akt pathway via cleaving DNA-PKcs. These multiple effects, associated with the regimen of cancer therapeutic strategies, make bromovanin very appealing for future development as a novel anticancer drug.


Assuntos
Apoptose , Benzaldeídos/química , Benzaldeídos/farmacologia , Dano ao DNA , Proteína Quinase Ativada por DNA/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , Humanos , Células Jurkat
15.
Int J Radiat Oncol Biol Phys ; 65(3): 842-50, 2006 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-16751065

RESUMO

PURPOSE: There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. METHODS AND MATERIALS: Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and gamma-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. RESULTS: The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and gammaH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy gamma-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G(2)/M arrest as compared with parental cells. In addition, the G(2)/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. CONCLUSION: HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS cancer patients to radiotherapy.


Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Reparo do DNA/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Produtos do Gene tat/fisiologia , Tolerância a Radiação , Linhagem Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Ensaio Cometa , Dano ao DNA/genética , Fase G2/genética , Fase G2/efeitos da radiação , Expressão Gênica , HIV-1 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Tolerância a Radiação/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana
16.
Int J Mol Med ; 16(3): 455-62, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16077955

RESUMO

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of a sub-family of phosphoinositol 3-kinases, has been reported overexpressed in various human cancers, but its significance is unclear. In the present study, we generated the stable cell line HeLa(siRNAH1) of silenced DNA-PKcs by transfecting HeLa cells with the siRNA construct targeting the catalytic motif of DNA-PKcs. The expression of DNA-PKcs was markedly suppressed in HeLa(siRNAH1) cells, and eventuating in increased cellular sensitivity to ionizing radiation as well as cisplatin. Microarray assay was used to explore the transcriptional profiling of signal transduction-associated genes. The results demonstrated that 15 genes were up-regulated and eight were down-regulated in HeLa(siRNAH1) as compared with the HeLa(control) cells that transfected with non-specific siRNA construct. Seven of the up-regulated genes are associated with the interferon-signaling events, the others function in the BMP signal pathway, or as regulators of cell cycle and differentiation. The down-regulated genes include IL8, IL10RA, DAPK3, and those involved in nuclear factor of activated T cells (NFAT) signal pathway and endocrine responsiveness. Using the NFAT-driving secreted alkaline phosphatase reporter expression system, we further confirmed that NFAT transcriptional activity was markedly minimized after silencing DNA-PKcs. These results demonstrated that inactivation of DNA-PKcs altered the transcriptional level of certain signal transduction-associated genes related to proliferation and differentiation.


Assuntos
Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Inativação Gênica , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Diferenciação Celular , Proliferação de Células , Proteína Quinase Ativada por DNA , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Vetores Genéticos/genética , Células HeLa , Humanos , Fatores de Transcrição NFATC , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transcrição Gênica/genética , Transfecção , Regulação para Cima/genética
17.
Zhonghua Zhong Liu Za Zhi ; 26(9): 521-4, 2004 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-15555279

RESUMO

OBJECTIVE: To study the effect of overexpression of Smad7 gene on cell proliferation in human bronchial epithelial cell lines. METHODS: Human bronchial epithelial cell lines, BEP2D and BERP35T2 cells, were cotransfected with the mammalian expression vectors PCISmad7.neo and pMyc-SEAP, the latter was ac-myc cis-acting enhancer element fused with alkaline phosphatase (SEAP) reporter gene. Expression of c-myc, p15 and p21 mRNA was detected by RT-PCR before and after stable transfection of Smad7 into BEP2D and BERP35T2 cells in order to study the regulation of TGF-beta-mediated growth inhibition. RESULTS: After BEP2D and BERP35T2 cells transfected with Smad7, the transcriptional activity of c-myc was significantly increased. Smad7 overexpressing cells showed upregulation of c-myc expression and downregulation of p15 and p21 expression, which contributed to the loss of TGF-beta responses in these cells. CONCLUSION: Overexpression of Smad7 may facilitate cell proliferation by antagonizing TGF-beta-mediated antiproliferative gene responses.


Assuntos
Brônquios/citologia , Proliferação de Células , Células Epiteliais/citologia , Proteína Smad7/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Transformação Celular Neoplásica , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Humanos , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Proteína Smad7/genética , Transfecção , Fator de Crescimento Transformador beta/genética
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