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1.
Anticancer Res ; 40(2): 813-823, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32014924

RESUMO

BACKGROUND/AIM: Olaparib was previously shown to synergistically enhance the cytotoxicity of DNA synthesis inhibitors in oesophageal carcinoma (OC) cell lines. However, the mechanisms of this synergy are not fully understood. As P53 binding protein 1 (53BP1) expression was previously shown to potentiate the anticancer effect of olaparib, we investigated the involvement of 53BP1 in the synergetic cytotoxic effects of olaparib and anticancer drugs in KYSE70 cells. MATERIALS AND METHODS: Experiments included small interfering RNA transfection, growth inhibition assays, western blots, immunofluorescence, and flow cytometry. RESULTS: The toxicity of DNA synthesis-inhibiting agents plus olaparib was decreased when 53BP1 was depleted. Olaparib cotreatment significantly increased phosphorylated H2A histone family member X (γH2AX) foci as well as 53BP1/γH2AX co-localisation in anticancer drug-treated cells. Silencing of 53BP1 suppressed anticancer drug-induced apoptosis with or without olaparib. CONCLUSION: Olaparib potentiates the cytotoxicity of anticancer drugs through 53BP1 in OC cells.


Assuntos
Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transfecção
2.
J Cancer Res Clin Oncol ; 146(3): 721-737, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31786739

RESUMO

PROPOSE: Poly (ADP-ribose) polymerase 1 inhibitors were originally investigated as anti-cancer therapeutics with BRCA1/2 genes mutation. Here, we investigate the effectiveness of a novel PARP1 inhibitor fluzoparib, for enhancing the radiation sensitivity of NSCLC cells lacking BRCA1/2 mutation. METHODS: We used MTS assays, western blotting, colony formation assays, immunofluorescence staining, and flow cytometry to evaluate the radiosensitization of NSCLC cells to fluzoparib and explore the underlying mechanisms in vitro. Through BRCA1 and RAD50 genes knockdown, we established dysfunctional homologous recombination (HR) DNA repair pathway models in NSCLC cells. We next investigated the radiosensitization effect of fluzoparib in vivo using human NSCLC xenograft models in mice. The expression of PARP1 and BRCA1 in human NSCLC tumor samples was measured by immunohistochemistry. Furthermore, we sequenced HR-related gene mutations and analyzed their frequencies in advanced NSCLC. RESULTS: In vitro experiments in NSCLC cell lines along with in vivo experiments using an NSCLC xenograft mouse model demonstrated the radiosensitization effect of fluzoparib. The underlying mechanisms involved increased apoptosis, cell-cycle arrest, enhanced irradiation-induced DNA damage, and delayed DNA-damage repair. Immunohistochemical staining showed no correlation between the expression of PARP1 and BRCA1. Moreover, our sequencing results revealed high mutation frequencies for the BRCA1/2, CHEK2, ATR, and RAD50 genes. CONCLUSION: The potential therapeutic value of fluzoparib for increasing the radiation sensitivity of NSCLC is well confirmed. Moreover, our findings of high mutation frequencies among HR genes suggest that PARP1 inhibition may be an effective treatment strategy for advanced non-small cell lung cancer patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína BRCA1 , Proteína BRCA2 , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Mutação , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Inorg Chem ; 58(23): 16279-16291, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31738050

RESUMO

Many clinical trials using combinations of platinum drugs and PARP-1 inhibitors (PARPi) have been carried out, with the hope that such combinations will lead to enhanced therapeutic outcomes against tumors. Herein, we obtained seven potential PARPi with structural diversity and then conjugated them with cisplatin-based platinum(IV) complexes. Both the synthesized PARPi ligands and PARPi-Pt conjugates [PARPi-Pt(IV)] show inhibitory effects against PARP-1's catalytic activity. The PARPi-Pt(IV) conjugates are cytotoxic in a panel of human cancer cell lines, and the leading ones display the ability to overcome cisplatin resistance. A mechanistic investigation reveals that the representative PARPi-Pt(IV) conjugates efficiently enter cells, bind to genomic DNA, disturb cell cycle distribution, and induce apoptotic cell death in both cisplatin-sensitive and -resistant cells. Our study provides a strategy to improve the cytotoxicity of platinum(IV)-based anticancer complexes and overcome cisplatin resistance by using a small-molecule anticancer complex that simultaneously damages DNA and inhibits PARP.


Assuntos
Antineoplásicos/farmacologia , Compostos Organoplatínicos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Relação Estrutura-Atividade
4.
Nucleic Acids Res ; 47(16): 8502-8520, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616951

RESUMO

Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Reparo do DNA , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Acetilação/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dano ao DNA , Células HEK293 , Humanos , Mutação , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
5.
Cancer Sci ; 110(11): 3543-3552, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541611

RESUMO

Poly ADP-ribose polymerase inhibitors (PARPi) have shown promising therapeutic efficacy in triple-negative breast cancer (TNBC) patients. However, resistance ultimately develops, preventing a curative effect from being attained. Extensive investigations have indicated the diversity in the mechanisms underlying the PARPi sensitivity of breast cancer. In this study, we found that DNA damage binding protein 2 (DDB2), a DNA damage-recognition factor, could protect TNBC cells from PARPi by regulating DNA double-strand break repair through the homologous recombination pathway, whereas the depletion of DDB2 sensitizes TNBC cells to PARPi. Furthermore, we found that DDB2 was able to stabilize Rad51 by physical association and disrupting its ubiquitination pathway-induced proteasomal degradation. These findings highlight an essential role of DDB2 in modulating homologous recombination pathway activity and suggest a promising therapeutic target for TNBC.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Proteínas de Neoplasias/metabolismo , Ubiquitinação
6.
Int J Mol Sci ; 20(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554189

RESUMO

Class III receptor tyrosine kinase (RTK) inhibitors targeting mainly FLT3 or c-KIT have not been well studied in lung cancer. To identify a small molecule potentially targeting class III RTK, we synthesized novel small molecule compounds and identified 5-(4-bromophenyl)-N-(naphthalen-1-yl) oxazol-2-amine (AIU2001) as a novel class III RKT inhibitor. In an in vitro kinase profiling assay, AIU2001 inhibited the activities of FLT3, mutated FLT3, FLT4, and c-KIT of class III RTK, and the proliferation of NSCLC cells in vitro and in vivo. AIU2001 induced DNA damage, reactive oxygen species (ROS) generation, and cell cycle arrest in the G2/M phase. Furthermore, AIU2001 suppressed the DNA damage repair genes, resulting in the 'BRCAness'/'DNA-PKness' phenotype. The mRNA expression level of STAT5 was downregulated by AIU2001 treatment and knockdown of STAT5 inhibited the DNA repair genes. Our results show that compared to either drug alone, the combination of AIU2001 with a poly (ADP-ribose) polymerase (PARP) inhibitor olaparib or irradiation showed synergistic efficacy in H1299 and A549 cells. Hence, our findings demonstrate that AIU2001 is a candidate therapeutic agent for NSCLC and combination therapies with AIU2001 and a PARP inhibitor or radiotherapy may be used to increase the therapeutic efficacy of AIU2001 due to inhibition of DNA damage repair.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares , Camundongos , Estrutura Molecular , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Oncol Rep ; 42(5): 2097-2107, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545455

RESUMO

Poly(ADP­ribose) polymerase (PARP) inhibitors have little effect on homologous recombination repair (HRR)­proficient tumor types, such as cervical cancer. In addition to catalytic activity, the PARP inhibitor, BMN673, traps PARP1 on damaged DNA and induces cytotoxic effects. The aim of the present study was to evaluate the therapeutic effect of PI3K inhibitors and BMN673 on cervical cancer cells. The Chou­Talalay method was used to assess the synergistic effect of drug combinations on cervical cancer cells. The effect of PI3K inhibitors and BMN673 on cell growth and survival were also assessed via a Cell Counting Kit­8 assay and three­dimensional sphere culture. Cell migration and invasion were assessed via Transwell migration and Matrigel invasion assays, respectively. In addition, DNA damage and HRR competency were assessed via immunofluorescent staining analysis of γH2AX and RAD51 foci, and tail moment in a comet assay. PARP1 binding in chromatin was assessed via a cellular trapping assay. Ex vivo cultured sections of patient­derived cervical tumors were subjected to drug exposure followed by histological and immunohistochemical analyses. The results revealed that the PI3K p110α inhibitor BYL719 and the PARP inhibitor BMN673 synergized to inhibit cervical cancer cell proliferation, migration and invasion in vitro and ex vivo. However, the pan­PI3K inhibitor BKM120 did not produce the aforementioned effects. Additionally, cervical cancer cells exhibiting aberrant PI3K activation were more responsive to the combined inhibition of PI3K p110α and PARP. Mechanistically, BYL719 co­operated with BMN673 to increase PARP1 trapping on chromatin, induce severe DNA damage and exert cytotoxic effects. The combined use of BMN673 and BYL719 may serve as a promising therapeutic strategy for patients with cervical cancer exhibiting aberrant PI3K activation.


Assuntos
Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Tiazóis/farmacologia , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico
8.
Exp Eye Res ; 188: 107790, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31494107

RESUMO

Oxidative stress (OxS) is involved in the development of cell injures occurring in retinal diseases while Poly(ADP-ribose) Polymerase-1 (PARP-1) is a key protein involved in the repair of the DNA damage caused by OxS. Inhibition of PARP-1 activity with the pharmacological inhibitor PJ34 in mouse retinal explants subjected to H2O2-induced oxidative damage resulted in an increase of apoptotic cells. Reduction of cell growth was also observed in the mouse cone like cell line 661 W in the presence of PJ34 under OxS conditions. Mass spectrometry-based phosphoproteomics analysis performed in 661 W cells determined that OxS induced significant changes in the phosphorylation in 1807 of the 8131 peptides initially detected. Blockade of PARP-1 activity after the oxidative treatment additionally increased the phosphorylation of multiple proteins, many of them at SQ motifs and related to the DNA-damage response (DDR). These motifs are substrates of the kinases ATM/ATR, which play a central role in DDR. Western blot analysis confirmed that the ATM/ATR activity measured and the phosphorylation at SQ motifs of ATM/ATR substrates was augmented when PARP-1 activity was inhibited under OxS conditions, in 661 W cells. Phosphorylation of ATM/ATR substrates, including the phosphorylation of the histone H2AX were also induced in organotypic cultures of retinal explants subjected to PARP-1 inhibition during exposure to OxS. In conclusion, inhibition of PARP-1 increased the phosphorylation and hence the activation of several proteins involved in the response to DNA damage, like the ATM protein kinase. This finally resulted in an augmented injury in mouse retinal cells suffering from OxS. Therefore, the inhibition of PARP-1 activity may have a negative outcome in the treatment of retinal diseases in which OxS is involved.


Assuntos
Dano ao DNA , Proteínas do Olho/metabolismo , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Retina/patologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Caspase 3/metabolismo , Morte Celular , Linhagem Celular , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Histonas/metabolismo , Peróxido de Hidrogênio/toxicidade , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Oxidantes/toxicidade , Fenantrenos/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Retina/metabolismo
9.
Int J Radiat Biol ; 95(12): 1597-1612, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31490091

RESUMO

Background and purpose: Poly(ADP-ribose)polymerase-1 (PARP1) and DNA-dependent protein kinase (DNA-PK) play key roles in the repair of radiation-induced DNA double strand breaks, but it is unclear which is the preferred therapeutic target in radiotherapy. Here we compare small molecule inhibitors of both as radiosensitizers of head and neck squamous cell carcinoma (HNSCC) cell lines.Methods: Two PARP1 inhibitors (olaparib, veliparib) and two DNA-PK inhibitors (KU57788, IC87361) were tested in 14 HNSCC cell lines and two non-tumorigenic lines (HEK-293 and WI-38/Va-13), with drug exposure for 6 or 24 h post-irradiation, using regrowth assays. For three lines (UT-SCC-54C, -74B, -76B), radiosensitization was also assessed by clonogenic assay under oxia and acute (6 h) anoxia, and for 54C cells under chronic hypoxia (0.2% O2 for 48 h). Relationships between sensitizer enhancement ratios (SER) and gene expression, assessed by RNA sequencing, were evaluated.Results: The inhibitors were minimally cytotoxic in the absence of radiation, with 74B and 54C cells the most sensitive to both olaparib and KU57788. Median SER values for each inhibitor at 1.1 µM were 1.12 (range 1.02-1.24) for olaparib, 1.08 (1.04-1.13) for veliparib, 1.35 (1.10-1.64) for IC87361 and 1.77 (1.41-2.38) for KU57788. The higher SER values for the DNA-PK inhibitors were observed with all cell lines (except HEK-293) and all concentrations tested and were confirmed by clonogenic assay. Radiosensitization by the DNA-PK inhibitors correlated with expression of SLFN11 mRNA. Radiosensitization by IC87361 and olaparib was significantly enhanced under acute anoxia and chronic hypoxia.Conclusions: The DNA-PK inhibitors KU57788 and IC87361 are more effective radiosensitizers than the PARP-1 inhibitors olaparib and veliparib at non-cytotoxic concentrations in HNSCC cell cultures and their activity is enhanced by SLFN11 and hypoxia.


Assuntos
Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Hipóxia Tumoral/efeitos dos fármacos , Hipóxia Tumoral/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo
10.
Breast Cancer Res ; 21(1): 104, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492187

RESUMO

BACKGROUND: Breast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence. METHODS: We evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients. RESULTS: Our results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival. CONCLUSIONS: Based on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.


Assuntos
Antineoplásicos/farmacologia , Recombinação Homóloga/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pirazinas/farmacologia , Pirazóis/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Rad51 Recombinase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
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
12.
Nat Commun ; 10(1): 2954, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273204

RESUMO

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cromatina/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Recombinação Homóloga/genética , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
13.
Phytother Res ; 33(9): 2347-2359, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273855

RESUMO

As yet, there was no effective pharmacological therapy approved for non-alcoholic fatty liver disease (NAFLD). Here, we aimed to evaluate the therapeutic potential of puerarin against NAFLD and explored the underlying mechanisms. C57BL/6J mice were fed with a high-fat high-sucrose (HFHS) diet with or without puerarin coadministration intragastrically. The levels of hepatocellular injury, steatosis, fibrosis, and mitochondrial and metabolism alteration were detected. First, puerarin ameliorated histopathologic abnormalities due to HFHS. We observed a marked increase in hepatic lipid content, inflammation, and fibrosis level, which were attenuated by puerarin. Possible mechanisms were related to puerarin-mediated activation of PI3K/AKT pathway and further improvement in fatty acid metabolism. Puerarin restored the NAD+ content and beneficially affected the hepatic mitochondrial function, which attenuated HFHS-induced steatosis and metabolic disturbances. Finally, hepatic PARP-1 was activated due to excessive fat intake. Puerarin attenuated the PARP-1 expression in HFHS-fed mice, and PJ34, the PARP inhibitor, could mimic these protections of puerarin. However, pharmacological inhibition of PI3K disabled the protection of puerarin or PJ34 toward NAD+ refilling and mitochondrial homeostasis. In conclusion, our findings indicated that puerarin could be a promising and practical therapeutic strategy in NAFLD through modulating PARP-1/PI3K/AKT signaling pathway and further facilitating mitochondrial function.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Isoflavonas/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sacarose/efeitos adversos , Vasodilatadores/uso terapêutico , Animais , Humanos , Isoflavonas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Vasodilatadores/farmacologia
14.
Nucleic Acids Res ; 47(16): 8563-8580, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31291457

RESUMO

Creating access to DNA double-strand break (DSB) sites in the chromatin context is an essential step during the repair process, but much remains to be determined about its regulatory mechanisms. Here, using a novel reporter cassette for simultaneous detection of homologous recombination (HR) and nonhomologous end joining (NHEJ) at the same chromosomal site, we report that the efficiency of HR but not NHEJ negatively correlates with nucleosome density. We demonstrate that PARP1 is required for HR by modulating nucleosome density at damage sites. Mechanistic studies indicate that the ATPase domain of BRG1 and the ZnF domain of SIRT1 interact with poly-ADP ribose (PAR) in response to DNA damage, and are responsible for bringing the two factors to broken DNA ends. At DNA damage sites, BRG1 and SIRT1 physically interact, whereupon SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, stimulating its ATPase activity to remodel chromatin and promote HR.


Assuntos
DNA Helicases/genética , DNA/genética , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Reparo de DNA por Recombinação , Sirtuína 1/genética , Fatores de Transcrição/genética , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Cloroquina/farmacologia , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismo
15.
Molecules ; 24(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261939

RESUMO

Breast cancer is considered the most common and deadly cancer among women worldwide. Nanomedicine has become extremely attractive in the field of cancer treatment. Due to the high surface to volume ratio and other unique properties, nanomaterials can be specifically targeted to certain cells and tissues to interact with the living systems. The strategic planning of this study is based on using the nanoprecipitation method to prepare nanoparticles BZP-NPs (3.8-5.7 nm) of the previously prepared benzofuran-pyrazole compound (IV) BZP which showed promising cytotoxic activity. The capacity of BZP and BZP-NPs to suppress the growth of human breast tumor MCF-7 and MDA-MB-231 cells was evaluated using MTT assay. The IC50 doses of BZP and BZP-NPs targeting normal breast cells MCF-12A exceeded those targeting the cancer cells by >1000-fold, demonstrating their reasonable safety profiles in normal cells. Furthermore, cell cycle analysis, apoptosis induction detection, assessment of p53, Bcl-2, caspase-3, and PARP-1 levels of BZP and its nano-sized-BZP-NPs particles were also evaluated. Although the obtained results were in the favor of compound IV in its normal-sized particles, BZP-NPs appeared as a hit compound which showed improved cytotoxicity against the tested human breast cancer cells associated with the induction of pre-G1 apoptosis as well as cell cycle arrest at G2/M phase. The increase in caspase-3 level, upregulation of p53, and downregulation of Bcl-2 protein expression levels confirmed apoptosis. Furthermore, ELISA results exhibited that BZP-NPs produced a more favorable impact as a PARP-1 enzyme inhibitor than the parent BZP.


Assuntos
Benzofuranos/síntese química , Neoplasias da Mama/enzimologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Pirazóis/síntese química , Benzofuranos/química , Benzofuranos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Difusão Dinâmica da Luz , Feminino , Humanos , Células MCF-7 , Microscopia Eletrônica de Transmissão , Nanopartículas , Tamanho da Partícula , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pirazóis/química , Pirazóis/farmacologia
16.
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
17.
Nucleic Acids Res ; 47(17): 9132-9143, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31329989

RESUMO

Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.


Assuntos
Proteína BRCA1/genética , DNA Glicosilases/genética , Neoplasias Ovarianas/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Oxirredução/efeitos dos fármacos , Ftalazinas/efeitos adversos , Ftalazinas/farmacologia , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Mutações Sintéticas Letais/genética
18.
Nature ; 572(7768): 254-259, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316209

RESUMO

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6-9-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.


Assuntos
Evasão da Resposta Imune , Leucemia Mieloide Aguda/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Evasão Tumoral , Animais , Antígenos CD34/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Ligantes , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Mol Sci ; 20(12)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242618

RESUMO

One of the most common malignancies in men is prostate cancer, for which androgen deprivation is the standard therapy. However, prostate cancer cells become insensitive to anti-androgen treatment and proceed to a castration-resistant state with limited therapeutic options. Therefore, besides the androgen deprivation approach, novel biomarkers are urgently required for specific targeting in this deadly disease. Recently, germline or somatic mutations in the homologous recombination (HR) DNA repair genes have been identified in at least 20-25% of metastatic castration-resistant prostate cancers (mCRPC). Defects in genes involved in HR DNA repair can sensitize cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors, a class of drugs already approved by the Food and Drug Administration (FDA) for breast and ovarian cancer carrying germline mutations in BRCA1/2 genes. For advanced prostate cancer carrying Breast cancer1/2 (BRCA1/2) or ataxia telengiectasia mutated (ATM) mutations, preclinical studies and clinical trials support the use of PARP-inhibitors, which received breakthrough therapy designation by the FDA. Based on these assumptions, several trials including DNA damage response and repair (DDR) targeting have been launched and are ongoing for prostate cancer. Here, we review the state-of-the-art potential biomarkers that could be predictive of cancer cell synthetic lethality with PARP inhibitors. The identification of key molecules that are affected in prostate cancer could be assayed in future clinical studies to better stratify prostate cancer patients who might benefit from target therapy.


Assuntos
Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias da Próstata/genética , Reparo de DNA por Recombinação , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos como Assunto , Dano ao DNA , Instabilidade Genômica , Humanos , Masculino , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Reprodutibilidade dos Testes , Mutações Sintéticas Letais/genética , Resultado do Tratamento
20.
Am J Vet Res ; 80(7): 663-669, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31246122

RESUMO

OBJECTIVE: To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. SAMPLE: 1,440 samples of PBMCs from 6 healthy research horses. PROCEDURES: From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA. RESULTS: Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.


Assuntos
Cavalos/sangue , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Doenças dos Cavalos/fisiopatologia , Técnicas In Vitro , Inflamação/fisiopatologia , Leucócitos Mononucleares/enzimologia , Lipopolissacarídeos/farmacologia
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