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1.
Chem Biol Interact ; 312: 108813, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494105

RESUMO

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival. YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus. The aim of this study is to evaluate the action of VP on RMS cell lines. This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment. In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.


Assuntos
Proliferação de Células/efeitos dos fármacos , Verteporfina/farmacologia , Linhagem Celular Tumoral , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Fatores de Transcrição/metabolismo
2.
Chem Biol Interact ; 311: 108798, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31433962

RESUMO

Natural products are a valuable source of anticancer agents, with many naturally derived compounds currently used in clinical and preclinical treatments. This study aims to investigate the antiproliferative activity and potential mechanism of action of the xanthoquinodin JBIR-99, isolated from fungi Parengyodontium album MEXU 30,054 and identified by single-crystal X-ray crystallography. Cytotoxicity of xanthoquinodin was evaluated in a panel of human cancer cells lines and CCD-112-CoN normal colon cells, using the sulforhodamine B assay. PC-3 prostate cancer cells were used in biochemical assays including cell cycle, mitochondrial transmembrane potential (MTP), reactive oxygen species (ROS) and caspase activity. Expression levels of apoptosis-pathway-related proteins were analyzed by Western blot. The in vivo toxicity of xanthoquinodin was determined using a zebrafish model. Xanthoquinodin showed cytotoxicity in all cancer cell lines but demonstrated relative selective potency against PC-3 cells with an IC50 1.7 µM. In CCD-112-CoN cells, xanthoquinodin was non-cytotoxic at 100 µM. In PC-3 cells, the compound induced loss of MTP, production of ROS, and cell cycle arrest in S phase. The expression and activity of caspase-3 was increased, which correlates with the upregulation of Cyt c, Bax, nuclear factor kappa-B (NF-κB) (p65) and IKKß, and downregulation of poly ADP ribose polymerase (PARP-1) and Bcl-2. Lastly, xanthoquinodin did not cause any visible developmental toxicity in zebrafish at 50 µM. These results demonstrate xanthoquinodin induces apoptosis in PC-3 prostate cancer cells by activation of both intrinsic and extrinsic apoptotic pathways. In addition, the non-toxic effect in vivo indicates that xanthoquinodin could be a useful lead in the development of a novel, anti-cancer agent that is selective for prostate cancer.


Assuntos
Apoptose/efeitos dos fármacos , Ascomicetos/química , Cromonas/farmacologia , Ascomicetos/metabolismo , Linhagem Celular Tumoral , Cromonas/química , Cristalografia por Raios X , Citocromos c/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Conformação Molecular , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
3.
Gynecol Oncol ; 155(1): 144-150, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31434613

RESUMO

OBJECTIVES: Cervical cancer (CC) remains a major health problem worldwide. Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutics in ovarian cancer. We explored the preclinical in vitro and in vivo activity of olaparib against multiple primary whole exome sequenced (WES) CC cells lines and xenografts. METHODS: Olaparib cell-cycle, apoptosis, homologous-recombination-deficiency (HRD), PARP trapping and cytotoxicity activity was evaluated against 9 primary CC cell lines in vitro. PARP and PAR expression were analyzed by Western blot assays. Finally, olaparib in vivo antitumor activity was tested against CC xenografts. RESULTS: While none of the cell lines demonstrated HRD, three out of 9 (33.3%) primary CC cell lines showed strong PARylation activity and demonstrated high sensitivity to olaparib in vitro treatment (cutoff IC50 values < 2 µM, p = 0.0012). Olaparib suppressed CC cell growth through cell cycle arrest in the G2/M phase and caused apoptosis (p < 0.0001). Olaparib activity in CC involved both PARP enzyme inhibition and trapping. In vivo, olaparib significantly impaired CC xenografts tumor growth (p = 0.0017) and increased overall animal survival (p = 0.008). CONCLUSIONS: A subset of CC primary cell lines is highly responsive to olaparib treatment in vitro and in vivo. High level of PARylation correlated with olaparib preclinical activity and may represent a useful biomarker for the identification of CC patients benefitting the most from PARPi.


Assuntos
Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/enzimologia , Adulto , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Camundongos SCID , Pessoa de Meia-Idade , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
4.
Nat Commun ; 10(1): 2910, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266951

RESUMO

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinases Ciclina-Dependentes/genética , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Zhongguo Zhong Yao Za Zhi ; 44(13): 2686-2690, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31359678

RESUMO

To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.


Assuntos
Apoptose , Cistanche/química , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Animais , Caspase 3/metabolismo , Etanol , Glucose , Estresse Oxidativo , Oxigênio , Células PC12 , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2/metabolismo
6.
Oncol Rep ; 42(4): 1467-1474, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31322269

RESUMO

With the increasing use of poly(ADP­ribose) polymerase (PARP) inhibitors in cancer therapy, understanding their resistance is an urgent research quest. Additionally, CHFR is an E3 ubiquitin ligase, recruited to double­strand breaks (DSBs) by PAR. Furthermore, ALC1 is a new oncogene involved in the invasion and metastasis of breast cancer. Moreover, PARylated PARP1 activates ALC1 at sites of DNA damage, yet the underlying mechanism remains unclear. Mass spectrometric analysis, western blot analysis and immunoprecipitation were performed to confirm the interaction between CHFR and ALC1 in the physiological condition. Deletion mutants of CHFR and ALC1 were generated to map the interaction domain. PARP1/2 inhibitors were added to identify the ubiquitination of ALC1 by CHFR. ALC1 half­life was examined to compare the expression of ALC1 protein in the presence and absence of PARP1/2 inhibitors. The results revealed that the transcriptional level of ALC1 was not upregulated in breast cancer tissues. CHFR interacted with ALC1. The PBZ domain of CHFR, the PMD domain and the MACRO domain of ALC1 domain are the necessary regions for the interaction depending on PAR. Ubiquitination of ALC1 by CHFR was dependent on PARylation and resulted in the degradation of PARylated ALC1. PARP1/2 inhibitors decreased the ubiquitination of PAR­dependent ALC1, and the expression of ALC1 was upregulated by PARP1/2 inhibitors. Ubiquitination mediated by CHFR resulted in the degradation of ALC1. In conclusion, PARP1/2 inhibitors decrease the ubiquitination of ALC1 leading to the accumulation of ALC1, which affects the therapeutic effects of DNA damage response drugs in breast cancer treatment.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Poli(ADP-Ribose) Polimerase-1/metabolismo , Transcrição Genética , Ubiquitinação/efeitos dos fármacos
7.
Eur J Med Chem ; 177: 338-349, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158748

RESUMO

A series of styrylquinolines was designed and synthesized based on the four main quinoline scaffolds including oxine, chloroxine and quinolines substituted with a hydroxyl group or chlorine atom at the C4 position. All of the compounds were tested for their anticancer activity on wild-type colon cancer cells (HCT 116) and those with a p53 deletion. Analysis of SAR revealed the importance of electron-withdrawing substituents in the styryl part and chelating properties in the quinoline ring. The compounds that were more active were also tested on a panel of four cancer cell lines with mutations in TP53 tumor suppressor gene. The results suggest that styrylquinolines induce cell cycle arrest and activate a p53-independent apoptosis. The apparent mechanism of action was studied for the most promising compounds, which produced reactive oxygen species and changed the cellular redox balance.


Assuntos
Antineoplásicos/farmacologia , Quinolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estirenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Heme Oxigenase-1/metabolismo , Humanos , Estrutura Molecular , Poli(ADP-Ribose) Polimerase-1/metabolismo , Quinolinas/síntese química , Quinolinas/química , Quinolinas/toxicidade , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Estirenos/síntese química , Estirenos/química , Estirenos/toxicidade , Proteína Supressora de Tumor p53/metabolismo
8.
Biosens Bioelectron ; 138: 111308, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31103013

RESUMO

Poly(ADP-ribose) polymerase-1 (PARP-1), as an original tumor marker, has aroused wide attention in recent years. However, only a few researches have been done for PARP-1 activity detection because PARP-1 is lack of optical or electrochemical property. In this work, a label-free and high-sensitive photoelectrochemical (PEC) biosensor for PARP-1 activity detection based on poly[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) has been designed. To the best of our knowledge, it is the first time that PEC has been used to monitor PARP-1 activity. PARP-1 were activated under the function of activated dsDNA, as a result, branched polymers of ADP-ribose (PAR) with plentiful negative charge were formed in the presence of nicotinamide adenine dinucleotide (NAD+). Subsequently, positively charged PFP with good photoelectrochemical properties, were absorbed on PAR via electrostatic interaction. High photocurrent was produced under light induction, which was depended on the PARP-1 activity. The biosensor has a wide linear range from 0.01 to 2 U with a detection limit of 0.007 U. The strategy has been applied in breast and ovarian cancer cells to detection PARP-1 activity with approving results, which signifies that it is a promising tool for clinical diagnosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Eletrodos , Ensaios Enzimáticos/métodos , Humanos , Indóis/farmacologia , Limite de Detecção , NAD/farmacologia , Processos Fotoquímicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Eletricidade Estática
9.
Comput Biol Chem ; 80: 314-323, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31078910

RESUMO

Inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) has turned out an innovative approach for cancer therapy due to its involvement in DNA repair pathways. Although several potent PARP-1 inhibitors have been identified, they exhibit high toxicity, resistivity and diverse pharmacological profile in clinical trials, which necessitate for extensive investigation and development of selective inhibitors. Therefore, the study aimed to identify selective natural PARP-1 inhibitors to reduce toxicity and resistivity with high potency. Accordingly, the combined approach of structure-based pharmacophore and molecular docking study was performed. Hence, the two hits (SN00167272 and STOCK1N-92279) were identified to have all the pharmacophoric features that showed interaction with key residues (Gly863, Ser904, Tyr896, and Tyr907) and least conserved residues (Tyr889 and Asp766). Additionally, these inhibitors represented interactions with unique selective residues (Asp756, Val762, Glu763 and Val886) and exhibited strong interaction with PARP-1 through binding free energy and molecular dynamics study. Hence, the identified hits could further considered for experimental investigations as they may reduce off-target and resistivity of currently available inhibitors and developed as potential anti-cancer agents in the future. Also, the study provides a specific structural insight which could further help to design selective and potent PARP-1 inhibitors.


Assuntos
Antineoplásicos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Poli(ADP-Ribose) Polimerase-1/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/toxicidade , Ligação Proteica , Estabilidade Proteica , Termodinâmica
10.
Oncol Rep ; 41(6): 3555-3564, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002368

RESUMO

Neoplastic transformation is characterized by metabolic rewiring to sustain the elevated biosynthetic demands of highly proliferative cancer cells. To obtain the precursors for macromolecule biosynthesis, cancer cells avidly uptake and metabolize glucose and glutamine. Thus, targeting the availability or metabolism of these nutrients is an attractive anticancer therapeutic strategy. To improve our knowledge concerning how cancer cells respond to nutrient withdrawal, the response to glutamine and/or glucose starvation was studied in human in vitro transformed fibroblasts, deeply characterized at the cellular and molecular level. Concomitant starvation of both nutrients led to rapid loss of cellular adhesion (~16 h after starvation), followed by cell death. Deprivation of glucose alone had the same effect, although at a later time (~48 h after starvation), suggesting that glucose plays a key role in enabling cell attachment to the extracellular matrix. Glutamine deprivation did not induce rapid cell death, but caused a prolonged arrest of cellular proliferation; the cells started dying only 96 h after starvation. Before massive cell death occurred, the effects of all the starvation conditions were reversible. Autophagy activation was observed in cells incubated in the absence of glucose for more than 48 h, while autophagy was not detected under the other starvation conditions. Markers of apoptotic cell death, such as caspase 3, caspase 9 and poly(ADP­ribose) polymerase 1 (PARP­1) proteolytic fragments, were not observed under any growth condition. Glucose and/or glutamine deprivation caused very rapid PARP­1 activation, with marked PARP­1 (poly­ADP) ribosylation and protein (poly­ADP) ribosylation. This activation was not due to starvation­induced DNA double­strand breaks, which appeared at the late stages of deprivation, when most cells died. Collectively, these results highlight a broad range of consequences of glucose and glutamine starvation, which may be taken into account when nutrient availability is used as a target for anticancer therapies.


Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Glucose/metabolismo , Glutamina/metabolismo , Apoptose/genética , Autofagia/genética , Caspases/genética , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Transformação Celular Neoplásica/metabolismo , Quebras de DNA de Cadeia Dupla , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucose/genética , Glutamina/genética , Humanos , Terapia de Alvo Molecular , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inanição/genética , Inanição/metabolismo
11.
Int J Oncol ; 54(4): 1466-1480, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30968148

RESUMO

It is well-known that the activation status of the P53, signal transducer and activator of transcription (Stat)3 and nuclear factor (NF)­κB signaling pathways determines the radiosensitivity of cancer cells. However, the function of these pathways in radiosensitive vs radioresistant cancer cells remains elusive. The present study demonstrated that adaptive expression of epidermal growth factor (EGF) following exposure to ionizing radiation (IR) may induce radiosensitization of pancreatic cancer (PC) cells through induction of the cyclin D1/P53/poly(ADP­ribose) polymerase pathway. By contrast, adaptively expressed interleukin (IL)­6 and insulin­like growth factor (IGF)­1 may promote radioresistance of PC cells, likely through activation of the Stat3 and NF­κB pathways. In addition, cyclin D1 and survivin, which are specifically expressed in the G1/S and G2/M phase of the cell cycle, respectively, are mutually exclusive in radiosensitive and radioresistant PC cells, while Bcl­2 and Bcl­xL expression does not differ between radiosensitive and radioresistant PC cells. Therefore, adaptively expressed EGF and IL­6/IGF­1 may alter these pathways to promote the radiosensitivity of PC cancers. The findings of the present study highlight potential makers for the evaluation of radiosensitivity and enable the development of effective regimens for cancer radiotherapy.


Assuntos
Carcinoma Ductal Pancreático/radioterapia , Fator de Crescimento Epidérmico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias Pancreáticas/radioterapia , Transdução de Sinais/efeitos da radiação , Apoptose/efeitos da radiação , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Humanos , Neoplasias Pancreáticas/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Tolerância a Radiação , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos da radiação
12.
Pharmazie ; 74(3): 142-146, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30961678

RESUMO

This study aimed to investigate the treatment effects and molecular mechanism of 3-aminobenzamide (3-AB) on intracranial aneurysms (IA). The IA model was established in male Sprague-Dawley (SD) rats and sham group was set up without ligation. The rats were intraperitoneally injected with normal saline in sham and model control groups and 10 mg/kg, 20 mg/kg and 40 mg/kg 3-AB for low, middle and high 3-AB groups for 3 months, respectively. The rates in and blood pressures of caudal artery were measured and anterior cerebral artery and olfactory artery were stained with hematoxylin and eosin (HE) for morphology observation. Besides, the effects of 3-AB on inflammatory cells, macrophages, neutrophils and T cells, were evaluated using immunohistochemistry. Gene expressions of TNF-α, MMP-9, MMP-2, iNOS, TLR4, PARP-1 and p65 were measured using qRT-PCR and the protein levels of TLR4, PARP-1 and p-p65 were evaluated using western blotting. Blood pressures of rats in 3-AB treatment groups were decreased in a dose-dependent manner. The damage of cerebral artery wall was alleviated and the inflammatory cells (macrophages, neutrophils and T cells) were reduced to some extent in 3-AB high-dose groups. The gene expression of TNF-α, MMP-9, MMP-2, iNOS, TLR4, PARP-1 and p65, as well as the protein expression of TLR4, PARP-1 and p-p65 in 3-AB treatment groups were decreased in a dose-dependent manner (P < 0.01).3-AB exhibited therapeutic effects on IA through inhibiting the secretions of inflammatory cytokines and MMPs.


Assuntos
Benzamidas/farmacologia , Doenças Arteriais Cerebrais/tratamento farmacológico , Aneurisma Intracraniano/tratamento farmacológico , Animais , Antígenos CD/metabolismo , Pressão Arterial , Doenças Arteriais Cerebrais/metabolismo , Doenças Arteriais Cerebrais/patologia , Doenças Arteriais Cerebrais/prevenção & controle , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Aneurisma Intracraniano/metabolismo , Aneurisma Intracraniano/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Mol Cell Biochem ; 457(1-2): 41-49, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30993494

RESUMO

In our previous study, we have shown that PARP-1 inhibition (genetic or pharmacological) ameliorates elastase-induced inflammation and emphysema. Since matrix metalloproteinases (MMPs) particularly MMP-2 and MMP-9 are known to play a critical role in emphysema development, the present work was designed to evaluate the effects of PARP-1 inhibition on their expression utilizing elastase-induced mouse model of emphysema. Our data show that olaparib administration at a dose of 5 mg/kg b.wt. (daily) significantly prevented the elastase-induced inflammation as indicated by decreased inflammatory cells particularly macrophages in BALF at 1 week post-injury. In addition, the drug restored the altered redox balance in the lungs of elastase-treated mice toward normal. Further, PCR data show that olaparib administration ameliorates the elastase-induced expression of MMP-2 and MMP-9 without having much effect on the expressions of their inhibitors TIMP-1 and TIMP-2. Next, our data on immunoblot, gelatin zymography, and immunohistochemical analysis indeed confirm that olaparib reduced the elastase-induced expression of MMP-2 and MMP-9. Reduction in the expression of metalloproteinases correlate well with the PARP activity as olaparib treatment suppressed the elastase-induced expression of PAR modified proteins markedly. Overall, our data strongly suggest that PARP-1 inhibition blunts elastase-induced MMP-2 and MMP-9 expression, which may be partly responsible for prevention of emphysema.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Elastase Pancreática/toxicidade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Enfisema Pulmonar/prevenção & controle , Animais , Modelos Animais de Doenças , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-2/biossíntese
14.
Chem Biol Interact ; 306: 110-116, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30991045

RESUMO

MicroRNAs (miRNAs) have been implicated in both biological and pathological processes in patients with systemic lupus erythematosus (SLE). Previous studies have demonstrated dysregulated expression of miR-199-3p, interleukin (IL)-10, and poly (ADP-ribose) polymerase-1 (PARP-1) in SLE. However, the underlying mechanisms of these aberrations have not been fully elucidated. In this study, we investigated the mechanism through which miR-199-3p dysregulation contributed to the pathogenesis of SLE. Altered gene expression was assessed by ChIP analysis. We then silenced the expression of candidate genes using siRNA for functional analysis; mRNA expression, protein levels, and protein expression were determined by qRT-PCR, ELISA, and western blotting, respectively. According to ChIP and qRT-PCR results, miR-199-3p was up-regulated in SLE patients. Moreover, IL-10 was found to be highly expressed in SLE patients by ELISA. Further, PARP1 was significantly down-regulated in SLE patients based on western blotting. Our results also indicated that miR-199-3p inhibits PARP1 expression by activating the ERK1/2 pathway, thereby increasing IL-10 expression. Significantly up-regulated miR-199-3p was inversely related to PARP-1 expression and positively correlated with IL-10 levels in SLE. As miR-199-3p was shown to target PARP-1 to activate the ERK1/2 pathway and promote IL-10 production, restoring physiological miR-199-3p levels could represent a potential therapeutic strategy for SLE treatment.


Assuntos
Interleucina-10/biossíntese , Lúpus Eritematoso Sistêmico/metabolismo , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Adulto , Feminino , Células HEK293 , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Masculino , Pessoa de Meia-Idade , Adulto Jovem
15.
Biol Pharm Bull ; 42(5): 758-763, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30842352

RESUMO

MicroRNAs were involved in a wide range of biological processes of diabetic nephropathy (DN). It is reported that miR-15b-5p was downregulated in the patients with DN. However, the mechanisms underlying the regulatory effects of miR-15b-5p on patients with diabetes remain unclear. Thus, this study aimed to investigate the role of miR-15b-5p during high glucose (HG)-induced apoptosis in human kidney cells. Quantitative real-time (qRT)-PCR was used to detect the level of miR-15b-5p. CCK-8 assay, EdU staining assays and flow cytometry were used to detect cell proliferation, apoptosis respectively in vitro. In addition, Western blotting was used to determine active caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), phosphorylated (p)-AKT, p-mammalian target of rapamycin (mTOR), p-S6, p-c-Jun N terminal kinase (JNK), p-p38 and p-extracellular signal-regulated kinase (ERK) proteins levels. The expression of miR-15b-5p in patients with DN were dramatically decreased compared with health persons. Similarly, HG down-regulated the expression of miR-15b-5p in HK-2 cells. In contrast, miR-15b-5p mimics alleviated HG-induced apoptosis in HK-2 cells via decreasing the expressions of active caspase 3 and cleaved PARP. EdU detection further confirmed that miR-15b-5p mimics attenuated the anti-proliferation effect of HG in HK-2 cells. Furthermore, HG-induced Akt/mTOR pathway downregulation and JNK upregulation were markedly reversed by miR-15b-5p mimics in cells. The data suggested that miR-15b-5p mimics protects HK-2 cells from HG-induced apoptosis. The anti-apoptotic effects of miR-15b-5p may due to the activation of the Akt/mTOR pathway as well as inactivation of JNK. Taken together, miR-15b-5p might be a potential therapeutic target for the treatment of patients with DN.


Assuntos
Materiais Biomiméticos/farmacologia , Glucose/farmacologia , MicroRNAs/química , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/química , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transfecção
16.
Nat Commun ; 10(1): 1307, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898999

RESUMO

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress in time. AF progression is driven by derailment of protein homeostasis, which ultimately causes contractile dysfunction of the atria. Here we report that tachypacing-induced functional loss of atrial cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. PARP1-mediated synthesis of ADP-ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces further DNA damage and contractile dysfunction. Accordingly, NAD+ replenishment or PARP1 depletion precludes functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in atrial cardiomyocytes and Drosophila. Consistently, cardiomyocytes of persistent AF patients show significant DNA damage, which correlates with PARP1 activity. The findings uncover a mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a possible therapeutic target that may preserve cardiomyocyte function in clinical AF.


Assuntos
Fibrilação Atrial/metabolismo , Fibrilação Atrial/prevenção & controle , Modelos Cardiovasculares , Miócitos Cardíacos/enzimologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Benzimidazóis/farmacologia , Células Cultivadas , Dano ao DNA , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Ativação Enzimática/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , Humanos , Larva/efeitos dos fármacos , Larva/metabolismo , Camundongos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/patologia , Niacinamida/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Marca-Passo Artificial/efeitos adversos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Pupa/efeitos dos fármacos , Pupa/metabolismo , Ratos , Ratos Wistar
17.
Mar Drugs ; 17(2)2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30821275

RESUMO

Lj-RGD3, which contains three Arg⁻Gly⁻Asp (RGD) motifs, was first identified from the buccal glands of Lampetra japonica and has been shown to suppress the tumor progression in the previous studies. Apart from the three RGD motifs, Lj-RGD3 is also characterized by its high content of histidine in its amino acid sequence. In order to clarify whether the histidine-rich characterization of Lj-RGD3 is also associated with its anti-tumor activity, mutants were designed in which the three RGD motifs (Lj-112), or all histidines (Lj-27) or both (Lj-26) were deleted. Furthermore, a mutant (Lj-42) in which all histidines and three RGD motifs were respectively substituted with alanines and three Ala⁻Gly⁻Asp (AGD) motifs, as well as a mutant (Lj-41) in which all histidines were substituted with alanines was synthesized to avoid alterations in structure which might further cause changes in the peptides' functions. After recombination and purification, recombinant Lj-112 (rLj-112), recombinant Lj-27 (rLj-27), recombinant Lj-41 (rLj-41), and recombinant Lj-RGD3 (rLj-RGD3) exhibited anti-proliferative activity in B16 cells, respectively; while recombinant Lj-26 (rLj-26) and recombinant Lj-42 (rLj-42) did not affect the proliferation of B16 cells significantly. In addition, the anti-proliferative activity of rLj-112 in B16 cells was due to apoptosis. Typical apoptosis features were observed, including chromatin condensation, fragmented DNA, and increased levels of cleaved caspase 3/caspase 7/nuclear enzyme poly (ADP-ribose) polymerase (PARP) in B16 cells. Similar to rLj-RGD3, rLj-112 was also capable of suppressing the migration and invasion of B16 cells by disturbing the F-actin arrangement. After labeling with FITC, rLj-112 was found localized in the cytoplasm of B16 cells, which induced the internalization of epidermal growth factor receptor (EGFR), suggesting that rLj-112 might block the EGFR mediated signaling pathway. Actually, the phosphorylation level of EGFR and its downstream signal molecules including Akt, PI3K, p38, and ERK1/2 was reduced in the rLj-112 treated B16 cells. In vivo, rLj-112 also inhibited the growth, weight, and volume of the tumors in B16 xenografted C57BL/6 mice without reducing their body weight, indicating that rLj-112 might be safe and might be used as an effective anti-tumor drug in the near future.


Assuntos
Receptores ErbB/metabolismo , Venenos de Peixe/genética , Venenos de Peixe/farmacologia , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Feminino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Commun ; 10(1): 1203, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867423

RESUMO

Vascular calcification is highly prevalent in end-stage renal diseases and is predictive of cardiovascular events and mortality. Poly(ADP-ribose) polymerase 1 (PARP1) inhibition or deletion is vasoprotective in several disease models. Here we show that PARP activity is increased in radial artery samples from patients with chronic renal failure, in arteries from uraemic rats, and in calcified vascular smooth muscle cells (VSMCs) in vitro. PARP1 deficiency blocks, whereas PARP1 overexpression exacerbates, the transdifferentiation of VSMCs from a contractile to an osteogenic phenotype, the expression of mineralization-regulating proteins, and calcium deposition. PARP1 promotes Runx2 expression, and Runx2 deficiency offsets the pro-calcifying effects of PARP1. Activated PARP1 suppresses miRNA-204 expression via the IL-6/STAT3 pathway and thus relieves the repression of its target, Runx2, resulting in increased Runx2 protein. Together, these results suggest that PARP1 counteracts vascular calcification and that therapeutic agents that influence PARP1 activity may be of benefit to treat vascular calcification.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Falência Renal Crônica/complicações , MicroRNAs/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Calcificação Vascular/patologia , Adenina/toxicidade , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Interleucina-6/metabolismo , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Artéria Radial/patologia , Ratos , Ratos Wistar , Fator de Transcrição STAT3/metabolismo , Técnicas de Cultura de Tecidos , Regulação para Cima , Uremia/induzido quimicamente , Uremia/complicações , Calcificação Vascular/etiologia
19.
Genes Dev ; 33(5-6): 310-332, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30804224

RESUMO

Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.


Assuntos
Apoptose , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Homeodomínio/metabolismo , Melanoma/genética , Melanoma/fisiopatologia , Mutação/genética , Fatores do Domínio POU/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Humanos , Autoantígeno Ku/metabolismo , Fatores do Domínio POU/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte Proteico
20.
Epigenetics Chromatin ; 12(1): 15, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777121

RESUMO

BACKGROUND: Recently, we showed that PARP1 is involved in cotranscriptional splicing, possibly by bridging chromatin to RNA and recruiting splicing factors. It also can influence alternative splicing decisions through the regulation of RNAPII elongation. In this study, we investigated the effect of PARP1-mediated chromatin changes on RNAPII movement, during transcription and alternative splicing. RESULTS: We show that RNAPII pauses at PARP1-chromatin structures within the gene body. Knockdown of PARP1 abolishes this RNAPII pausing, suggesting that PARP1 may regulate RNAPII elongation. Additionally, PARP1 alters nucleosome deposition and histone post-translational modifications at specific exon-intron boundaries, thereby affecting RNAPII movement. Lastly, genome-wide analyses confirmed that PARP1 influences changes in RNAPII elongation by either reducing or increasing the rate of RNAPII elongation depending on the chromatin context. CONCLUSIONS: These studies suggest a context-specific effect of PARP1-chromatin binding on RNA polymerase movement and provide a platform to delineate PARP1's role in RNA biogenesis and processing.


Assuntos
Montagem e Desmontagem da Cromatina , Processamento de RNA , Elongação da Transcrição Genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Polimerase II/metabolismo
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