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1.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118711, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32224192

RESUMO

Cardiotoxicity is a highly relevant, because often life-threatening, adverse effect of doxorubicin (Doxo)-based anticancer therapy. Here, we investigated the Doxo-response of cardiovascular stem/progenitor cells employing a mouse embryonic stem cell (mESC)-based in vitro differentiation model. Endothelial progenitor cells revealed a pronounced Doxo sensitivity as compared to mESC, differentiated endothelial-like (EC) and cardiomyocyte-like cells (CM) and CM progenitors, which rests on the activation of senescence. Doxo treatment of EC progenitors altered protein expression of individual endothelial markers, actin cytoskeleton morphology, mRNA expression of genes related to mitochondrial functions, autophagy, apoptosis, and DNA repair as well as mitochondrial DNA content, respiration and ATP production in the surviving differentiated EC progeny. By contrast, LDL uptake, ATP-stimulated Ca2+ release, and cytokine-stimulated ICAM-1 expression remained unaffected by the anthracycline treatment. Thus, exposure of EC progenitors to Doxo elicits isolated and persistent dysfunctions in the surviving EC progeny. In conclusion, we suggest that Doxo-induced injury of EC progenitors adds to anthracycline-induced cardiotoxicity, making this cell-type a preferential target for pharmacoprotective and regenerative strategies.

2.
Biochim Biophys Acta Mol Cell Res ; 1867(6): 118678, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32061892

RESUMO

The endothelium contributes to the pathophysiology of adverse effects caused by conventional (genotoxic) anticancer therapeutics (cAT). The relevance of structurally different types of cAT-induced DNA lesions for eliciting selected endothelial stress responses is largely unknown. Here, we analyzed the cAT-induced formation of DNA double-strand breaks (DSB), transcription blockage and DNA damage response (DDR) in time kinetic analyses employing a monolayer of primary human endothelial cells (HUVEC). We observed that the degree of cAT-induced transcription blockage, the number of DSB and activation of DDR-related factors diverge. For instance, ionizing radiation caused the formation of numerous DSB and triggerd a substantial activation of ATM/Chk2 signaling, which however were not accompanied by a significant transcription inhibition. By contrast, the DNA cross-linking cAT cisplatin triggered a rapid and substantial blockage of transcription, which yet was not reflected by an appreciable number of DSB or increased levels of pATM/pChk2. In general, cAT-stimulated ATM-dependent phosphorylation of Kap1 (Ser824) and p53 (Ser15) reflected best cAT-induced transcription blockage. In conclusion, cAT-induced formation of DSB and profound activation of prototypical DDR factors is independent of the inhibition of RNA polymerase II-regulated transcription in an endothelial monolayer. We suggest that DSB formed directly or indirectly following cAT-treatment do not act as comprehensive triggers of superior signaling pathways shutting-down transcription while, at the same time, causing an appreciable stimulation of the DDR. Rather, it appears that distinct cAT-induced DNA lesions elicit diverging signaling pathways, which separately control transcription vs. DDR activity in the endothelium.

3.
Bioorg Med Chem ; 28(4): 115279, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31980363

RESUMO

Natural compounds (NC) are an important source of anticancer drugs. The genomic DNA of tumor cells is a major target of conventional anticancer therapeutics (cAT). DNA damage elicits a complex stress response programme termed DNA damage response (DDR), with the PI3-like kinase ATM and ATR being the key regulators. Since the DDR coordinates mechanisms of DNA repair and apoptosis, hence regulating the balance between death and survival, it is an attractive target of novel anticancer strategies. The aim of the study was to identify natural compounds derived from endophytic fungi, lichens, marine sponges or plants that interfere with mechanisms of the DDR. To this end, the cytotoxic and DDR modulating potency of 296 natural compounds, used alone or in combination with the cAT cisplatin (Cis) and doxorubicin (Doxo) was investigated by fluorescence-based analysis of the ATM/ATR-catalyzed S139 phosphorylation of histone 2AX (γH2AX), a surrogate marker of DNA damage-triggered DDR. After initial screening, a total of ten natural compounds were identified that were toxic in pancreatic carcinoma cells and activated the DDR on their own and/or promoted the DDR if used in combination with cAT. Their mode of action was shown to be independent of drug transport mechanisms. Based on their chemical structures, DDR modulatory activity and published data we suggest the marine NC 5-epi-nakijiquinone Q and 5-epi-ilimaquinone as well as the fungal compound secalonic acid F as most promising NC-based drug candidates for future synthesis of DDR-modulating chemical derivatives and their preclinical in vitro and in vivo testing.

4.
Sci Rep ; 9(1): 13800, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551436

RESUMO

The endothelium represents the inner cell layer of blood vessels and is supported by smooth muscle cells and pericytes, which form the vessel structure. The endothelium is involved in the pathogenesis of many diseases, including the development of atherosclerosis. Due to direct blood contact, the blood vessel endothelium is inevitably exposed to genotoxic substances that are systemically taken up by the body, including benzo[a]pyrene, which is a major genotoxic component in cigarette smoke and a common environmental mutagen and human carcinogen. Here, we evaluated the impact of benzo[a]pyrene diol epoxide (BPDE), which is the reactive metabolite of benzo[a]pyrene, on the three innermost vessel cell types. Primary human endothelial cells (HUVEC), primary human smooth muscle cells (HUASMC) and primary human pericytes (HPC) were treated with BPDE, and analyses of cytotoxicity, cellular senescence and genotoxic effects were then performed. The results showed that HUVEC were more sensitive to the cytotoxic activity of BPDE than HUASMC and HPC. We further show that HUVEC display a detraction in the repair of BPDE-induced adducts, as determined through the comet assay and the quantification of BPDE adducts in post-labelling experiments. A screening for DNA repair factors revealed that the nucleotide excision repair (NER) proteins ERCC1, XPF and ligase I were expressed at lower levels in HUVEC compared with HUASMC and HPC, which corresponds with the impaired NER-mediated removal of BPDE adducts from DNA. Taken together, the data revealed that HUVEC exhibit an unexpected DNA repair-impaired phenotype, which has implications on the response of the endothelium to genotoxicants that induce bulky DNA lesions, including the development of vascular diseases resulting from smoking and environmental pollution.

5.
Purinergic Signal ; 15(3): 287-298, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31270713

RESUMO

Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19 µM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250 µM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.


Assuntos
Descoberta de Drogas , Naftalenos/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y2/efeitos dos fármacos , Animais , Humanos , Naftalenos/química , Antagonistas do Receptor Purinérgico P2Y/química , Suramina/análogos & derivados
6.
Biochem Pharmacol ; 164: 82-93, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936017

RESUMO

Cardiotoxicity is the dose limiting adverse effect of anthracycline-based anticancer therapy. Inhibitor studies point to Rac1 as therapeutic target to prevent anthracycline-induced cardiotoxicity. Yet, supporting genetic evidence is still missing and the pathophysiological relevance of different cardiac cell types is unclear. Here, we employed a tamoxifen-inducible cardiomyocyte-specific rac1 knock-out mouse model (Rac1flox/flox/MHC-MerCreMer) to investigate the impact of Rac1 expression in cardiomyocytes on cardiac injury following doxorubicin treatment. Distinctive stress responses resulting from doxorubicin treatment were observed, including upregulation of systemic markers of inflammation (IL-6, IL-1α, MCP-1), cardiac damage (ANP, BNP), DNA damage (i.e. DNA double-strand breaks (DSB)), DNA damage response (DDR) and cell death. Measuring the acute doxorubicin response, the serum level of MCP-1 was elevated, cardiac mRNA expression of Hsp70 was reduced and cardiac DDR was specifically enhanced in Rac1 deficient mice. The frequency of apoptotic heart cells remained unaffected by Rac1. Employing a subactue model, the number of doxorubicin-induced DSB was significantly reduced if Rac1 is absent. Yet, the doxorubicin-triggered increase in serum ANP and BNP levels remained unaffected by Rac1. Overall, knock-out of rac1 in cardiomyocytes confers partial protection against doxorubicin-induced cardiac injury. Hence, the data provide first genetic evidence supporting the view that pharmacological targeting of Rac1 is useful to widen the therapeutic window of anthracycline-based anticancer therapy by alleviating acute/subacute cardiomyocyte damage. Furthermore, considering published data obtained from the use of pharmacological Rac1 inhibitors, the results of our study indicate that Rac1-regulated functions of cardiac cell types others than cardiomyocytes additionally influence the adverse outcomes of anthracycline treatment on the heart.


Assuntos
Antraciclinas/toxicidade , Cardiopatias/induzido quimicamente , Cardiopatias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Neuropeptídeos/biossíntese , Proteínas rac1 de Ligação ao GTP/biossíntese , Animais , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuropeptídeos/genética , Proteínas rac1 de Ligação ao GTP/genética
7.
DNA Repair (Amst) ; 73: 17-27, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30413344

RESUMO

The accelerated ageing disease Werner Syndrome (WRN) is characterized by pronounced atherosclerosis. Here, we investigated the influence of WRN downregulation on the functionality of non-replicating human endothelial cells. RNAi-mediated downregulation of WRN reduces cell motility and enhances the expression of factors regulating adhesion, inflammation, hemostasis and vasomotor tone. Moreover, WRN influences endothelial barrier function and Ca2+-release, while cell adhesion, Dil-acLDL-uptake and the mRNA expression of NO-synthases (eNOS, iNOS) remained unaffected. Regarding motility, we propose that WRN affects Rac1/FAK/ß1-integrin-related mechanisms regulating cell polarity and directed motility. Since oxidative DNA base damage contributes to aging and atherosclerosis and WRN affects DNA repair, we investigated whether downregulation of base excision repair (BER) factors mimics the effects of WRN knock-down. Indeed, downregulation of particular WRN-interacting base excision repair (BER) proteins (APE1, NEIL1, PARP1) imitates the inhibitory effect of WRN on motility. Knock-down of OGG1, which does not interact with WRN, does not influence motility but increases the mRNA expression of E-selectin, ICAM, VCAM, CCL2 and VEGFR and stimulates adhesion. Thus, individual BER factors themselves differently impact endothelial cell functionality and homeostasis. Impairment of endothelial activities caused by genotoxic stressor (tBHQ) remained largely unaffected by WRN. Summarizing, both WRN, WRN-associated BER proteins and OGG1 promote the maintenance of endothelial cell homeostasis, thereby counteracting the development of ageing-related endothelial malfunction in non-proliferating endothelial cells.


Assuntos
DNA Helicases/metabolismo , Reparo do DNA , Homeostase , Células Endoteliais da Veia Umbilical Humana/metabolismo , Síndrome de Werner/enzimologia , Cálcio/metabolismo , Adesão Celular , Movimento Celular , Expressão Gênica , Humanos , Lipoproteínas LDL/metabolismo , Transporte Proteico
8.
Biochim Biophys Acta Mol Cell Res ; 1865(9): 1277-1292, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29932987

RESUMO

BACKGROUND: The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis. METHODS: We employed a constitutive Alb-Cre-driven rac1 knock-out and a poly I:C-inducible Mx1-Cre-based knock-out model and analyzed cytokine expression profiles in liver and other organs under basal situation and following LPS-induced endotoxemia by flow cytometry, qRT-PCR and immunocytochemistry. RESULTS: Constitutive Alb-Cre-driven rac1 knockout in hepatocytes altered the basal distribution and activation of immune cells in the liver and likewise in kidney and lung. Early systemic alterations in cytokine serum levels following LPS treatment remained unaffected by Rac1. Furthermore, lack of Rac1 in hepatocytes of untreated animals shifted the liver to a chronic inflammatory state, as depicted by an enhanced mRNA expression of marker genes related to activated macrophages. Upon acute LPS-induced endotoxemia, increased IL-10 mRNA expression in the liver of Alb-Cre Rac1-deficient mice provided an anti-inflammatory response. Employing a poly I:C-inducible Mx1-Cre-based rac1 knock-out, which allows a more widespread rac1 deletion in both hepatocytes and non-hepatocytes, we observed substantial differences regarding both basal and LPS-stimulated cytokine expression profiles as compared to the Alb-Cre system. CONCLUSIONS: Rac1-dependent mechanisms in hepatocytes and non-hepatocytes contribute to the maintenance of liver immune homeostasis under basal situation and following LPS-induced endotoxemia. Disturbed Rac1-regulated hepatocyte functions may promote liver damage under pathophysiological situation involving inflammatory stress.


Assuntos
Endotoxemia/enzimologia , Interleucina-10/genética , Lipopolissacarídeos/efeitos adversos , Fígado/imunologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Endotoxemia/imunologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Imunidade , Rim/imunologia , Fígado/enzimologia , Pulmão/imunologia , Macrófagos/metabolismo , Camundongos , Transdução de Sinais
9.
Cancer Lett ; 430: 34-46, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-29753759

RESUMO

Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3ß protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Meduloblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caenorhabditis elegans , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Testes de Toxicidade , Ureia/análogos & derivados , Ureia/farmacologia , Ureia/uso terapêutico
10.
Arch Toxicol ; 92(1): 441-453, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28710503

RESUMO

The anticancer efficacy of anthracyclines is limited by congestive heart failure. Clinically established markers of early onset of cardiotoxicity following anthracycline treatment and preventive measures are missing. Although statins are reported to alleviate anthracycline-induced cardiotoxicity in vivo, the molecular mechanisms involved remain elusive. In vitro data point to Rac1 as major target of the cytoprotective statin effects. Here we investigated whether specific inhibition of Rac1 by NSC23766 is as effective as lovastatin in preventing subacute cardiotoxicity following doxorubicin treatment. C57BL/6 mice were treated over 3 weeks with multiple low doses of doxorubicin (6 × 3 mg/kg BW, i.p.) and the level of DNA damage, apoptosis and regenerative proliferation as well as pro-inflammatory, pro-fibrotic and oxidative stress responses were investigated. Moreover, heart function was monitored by echocardiography. Doxorubicin induced subacute cardiotoxicity which was reflected on the level of residual DNA damage, frequency of apoptotic and mitotic cells as well as elevated mRNA expression of markers of heart failure, remodeling and mitochondrial biogenesis. These molecular markers of cardiotoxicity were mitigated to a similar extent by co-treatment with either lovastatin (10 mg/kg BW, p.o.) or NSC23766 (5 mg/kg BW, i.p.) three times a week. Moreover, doxorubicin caused diastolic dysfunction as reflected by increased E-wave acceleration time (EAT), which again was prevented by pharmacological inhibition of Rac1. Inhibition of Rac1 signaling is of major relevance for the cardioprotective effects of lovastatin in the context of anthracycline-induced cardiotoxicity. Moreover, EAT is a useful marker of subacute cardiotoxicity caused by persisting harmful effects of doxorubicin.


Assuntos
Cardiotoxicidade/etiologia , Doxorrubicina/efeitos adversos , Lovastatina/farmacologia , Neuropeptídeos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Aminoquinolinas/farmacologia , Animais , Antibióticos Antineoplásicos/efeitos adversos , Cardiotônicos/farmacologia , Cardiotoxicidade/fisiopatologia , Cardiotoxicidade/prevenção & controle , Dano ao DNA/efeitos dos fármacos , Diástole/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Eletrocardiografia , Masculino , Camundongos Endogâmicos C57BL , Neuropeptídeos/antagonistas & inibidores , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade Subaguda/métodos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores
11.
Cell Death Dis ; 8(8): e2978, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28796249

RESUMO

Thoracic radiotherapy causes damage of normal lung tissue, which limits the cumulative radiation dose and, hence, confines the anticancer efficacy of radiotherapy and impacts the quality of life of tumor patients. Ras-homologous (Rho) small GTPases regulate multiple stress responses and cell death. Therefore, we investigated whether pharmacological targeting of Rho signaling by the HMG-CoA-reductase inhibitor lovastatin influences ionizing radiation (IR)-induced toxicity in primary human lung fibroblasts, lung epithelial and lung microvascular endothelial cells in vitro and subchronic mouse lung tissue damage following hypo-fractionated irradiation (4x4 Gy). The statin improved the repair of radiation-induced DNA double-strand breaks (DSBs) in all cell types and, moreover, protected lung endothelial cells from IR-induced caspase-dependent apoptosis, likely involving p53-regulated mechanisms. Under the in vivo situation, treatment with lovastatin or the Rac1-specific small molecule inhibitor EHT1864 attenuated the IR-induced increase in breathing frequency and reduced the percentage of γH2AX and 53BP1-positive cells. This indicates that inhibition of Rac1 signaling lowers IR-induced residual DNA damage by promoting DNA repair. Moreover, lovastatin and EHT1864 protected lung tissue from IR-triggered apoptosis and mitigated the IR-stimulated increase in regenerative proliferation. Our data document beneficial anti-apoptotic and genoprotective effects of pharmacological targeting of Rho signaling following hypo-fractionated irradiation of lung cells in vitro and in vivo. Rac1-targeting drugs might be particular useful for supportive care in radiation oncology and, moreover, applicable to improve the anticancer efficacy of radiotherapy by widening the therapeutic window of thoracic radiation exposure.


Assuntos
Apoptose/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Lovastatina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Pironas/farmacologia , Qualidade de Vida , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismo
12.
Cell Death Dis ; 8(1): e2564, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28102848

RESUMO

Cancer patients receiving anthracycline-based chemotherapy are at risk to develop life-threatening chronic cardiotoxicity with the pathophysiological mechanism of action not fully understood. Besides the most common hypothesis that anthracycline-induced congestive heart failure (CHF) is mainly caused by generation of reactive oxygen species, recent data point to a critical role of topoisomerase II beta (TOP2B), which is a primary target of anthracycline poisoning, in the pathophysiology of CHF. As the use of the only clinically approved cardioprotectant dexrazoxane has been limited by the FDA in 2011, there is an urgent need for alternative cardioprotective measures. Statins are anti-inflammatory and anti-oxidative drugs that are clinically well established for the prevention of cardiovascular diseases. They exhibit pleiotropic beneficial properties beyond cholesterol-lowering effects that most likely rest on the indirect inhibition of small Ras homologous (Rho) GTPases. The Rho GTPase Rac1 has been shown to be a major factor in the regulation of the pro-oxidative NADPH oxidase as well as in the regulation of type II topoisomerase. Both are discussed to play an important role in the pathophysiology of anthracycline-induced CHF. Therefore, off-label use of statins or novel Rac1 inhibitors might represent a promising pharmacological approach to gain control over chronic cardiotoxicity by interfering with key mechanisms of anthracycline-induced cardiomyocyte cell death.


Assuntos
DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Insuficiência Cardíaca/genética , Neoplasias/complicações , Proteínas rac1 de Ligação ao GTP/genética , Antraciclinas/efeitos adversos , Antraciclinas/uso terapêutico , Morte Celular/efeitos dos fármacos , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/patologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NADPH Oxidases/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores
13.
J Clin Immunol ; 36(7): 684-92, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27473539

RESUMO

Hereditary defects in several genes have been shown to disturb the normal immune response to EBV and to give rise to severe EBV-induced lymphoproliferation in the recent years. Nevertheless, in many patients, the molecular basis of fatal EBV infection still remains unclear. The Fanconi anemia-associated protein 24 (FAAP24) plays a dual role in DNA repair. By association with FANCM as component of the FA core complex, it recruits the FA core complex to damaged DNA. Additionally, FAAP24 has been shown to evoke ATR-mediated checkpoint responses independently of the FA core complex. By whole exome sequencing, we identified a homozygous missense mutation in the FAAP24 gene (cC635T, pT212M) in two siblings of a consanguineous Turkish family who died from an EBV-associated lymphoproliferative disease after infection with a variant EBV strain, expressing a previously unknown EBNA2 allele.In order to analyze the functionality of the variant FAAP24 allele, we used herpes virus saimiri-transformed patient T cells to test endogenous cellular FAAP24 functions that are known to be important in DNA damage control. We saw an impaired FANCD2 monoubiquitination as well as delayed checkpoint responses, especially affecting CHK1 phosphorylation in patient samples in comparison to healthy controls. The phenotype of this FAAP24 mutation might have been further accelerated by an EBV strain that harbors an EBNA2 allele with enhanced activities compared to the prototype laboratory strain B95.8. This is the first report of an FAAP24 loss of function mutation found in human patients with EBV-associated lymphoproliferation.


Assuntos
Proteínas de Ligação a DNA/genética , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Mutação , Irmãos , Substituição de Aminoácidos , Ciclo Celular , Códon , Consanguinidade , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi , Evolução Fatal , Feminino , Genótipo , Homozigoto , Humanos , Contagem de Linfócitos , Transtornos Linfoproliferativos/virologia , Masculino , Linhagem , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Troca de Cromátide Irmã , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitinação , Sequenciamento Completo do Exoma
14.
Oncotarget ; 7(27): 41320-41335, 2016 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-27191498

RESUMO

Cisplatin (CisPt) is frequently used in the therapy of urothelial carcinoma (UC). Its therapeutic efficacy is limited by inherent or acquired drug resistance. Here, we comparatively investigated the CisPt-induced response of two different parental urothelial carcinoma cell lines (RT-112, J-82) with that of respective drug resistant variants (RT-112R, J-82R) obtained upon month-long CisPt selection. Parental RT-112 cells were ~2.5 fold more resistant to CisPt than J-82 cells and showed a different expression pattern of CisPt-related resistance factors. CisPt resistant RT-112R and J-82R variants revealed a 2-3-fold increased CisPt resistance as compared to their corresponding parental counterparts. Acquired CisPt resistance was accompanied by morphological alterations resembling epithelial mesenchymal transition (EMT). RT-112R cells revealed lower apoptotic frequency and more pronounced G2/M arrest following CisPt exposure than RT-112 cells, whereas no differences in death induction were observed between J-82 and J-82R cells. CisPt resistant J-82R cells however were characterized by a reduced formation of CisPt-induced DNA damage and related DNA damage response (DDR) as compared to J-82 cells. Such difference was not observed between RT-112R and RT-112 cells. J-82R cells showed an enhanced sensitivity to pharmacological inhibition of checkpoint kinase 1 (Chk1) and, moreover, could be re-sensitized to CisPt upon Chk1 inhibition. Based on the data we suggest that mechanisms of acquired CisPt resistance of individual UC cells are substantially different, with apoptosis- and DDR-related mechanisms being of particular relevance. Moreover, the findings indicate that targeting of Chk1 might be useful to overcome acquired CisPt resistance of certain subtypes of UC.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/genética , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/patologia , Urotélio/efeitos dos fármacos , Urotélio/metabolismo , Urotélio/patologia
15.
Oncotarget ; 7(24): 35832-35842, 2016 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-27127883

RESUMO

Patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) show better survival rates than those with HPV-negative HNSCC. While an enhanced radiosensitivity of HPV-positive tumors is clearly evident from single modality treatment, cisplatin is never administered as monotherapy and therefore its contribution to the enhanced cure rates of HPV-positive HNSCC is not known. Both cisplatin and radiotherapy can cause severe irreversible side effects and therefore various clinical studies are currently testing deintensified regimes for patients with HPV-positive HNSCC. One strategy is to omit cisplatin-based chemotherapy or replace it by less toxic treatments but the risk assessment of these approaches remains difficult. In this study we have compared the cytotoxic effects of cisplatin in a panel of HPV-positive and -negative HNSCC cell lines alone and when combined with radiation.While cisplatin-treated HPV-positive strains showed a slightly stronger inhibition of proliferation, there was no difference regarding colony formation. Cellular responses to the drug, namely cell cycle distribution, apoptosis and γH2AX-induction did not differ between the two entities but assessment of cisplatin-DNA-adducts suggests differences regarding the mechanisms that determine cisplatin sensitivity. Combining cisplatin with radiation, we generally observed an additive but only in a minority of strains from both entities a clear synergistic effect on colony formation. In summary, HPV-positive and -negative HNSCC cells were equally sensitive to cisplatin. Therefore replacing cisplatin may be feasible but the substituting agent should be of similar efficacy in order not to jeopardize the high cure rates for HPV-positive HNSCC.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Papillomaviridae/crescimento & desenvolvimento , Antineoplásicos/farmacologia , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos da radiação , Humanos , Papillomaviridae/fisiologia , Tolerância a Radiação/efeitos dos fármacos
16.
Biochim Biophys Acta ; 1863(8): 1969-78, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27108184

RESUMO

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.


Assuntos
Carcinoma de Células Escamosas/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Integrina alfaVbeta3/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Neoplasias da Língua/patologia , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cisplatino/farmacologia , Dasatinibe/farmacologia , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Genes src , Humanos , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Cadeias beta de Integrinas/fisiologia , Mitomicina/farmacologia , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Mutação Puntual , Multimerização Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Interferência de RNA , Neoplasias da Língua/genética , Neoplasias da Língua/metabolismo
17.
J Crohns Colitis ; 10(10): 1132-43, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27112707

RESUMO

BACKGROUND AND AIMS: The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD]. METHODS: Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potential toxicity, and immunosuppression. Activation of the Vav1/Rac1 pathway in lymphocytes was studied in IBD patients and in lamina propria immune cells in the presence or absence of thiopurine-analogues. RESULTS: Several thiopurine-analogues induced significantly higher T cell apoptosis than 6-mercaptopurine. We identified a compound, denoted B-0N, based on its capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. B-0N-treatment resulted in accelerated inhibition of Rac1 activity in primary peripheral blood T cells as well as in intestinal lamina propria immune cells. Compared with 6-thio-GTP and 6-mercaptopurine, B-0N-treatment was associated with decreased myelo- and hepatotoxicity. CONCLUSIONS: The Vav1/Rac1 pathway is activated in mucosal immune cells in IBD. The designer thiopurine-analogue B-0N induces immunosuppression more potently than 6-mercaptopurine.


Assuntos
Drogas Desenhadas/farmacologia , Imunossupressores/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Mercaptopurina/análogos & derivados , Mercaptopurina/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Estudos de Casos e Controles , Drogas Desenhadas/uso terapêutico , Desenho de Fármacos , Humanos , Imunossupressores/uso terapêutico , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mercaptopurina/uso terapêutico , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
Biochim Biophys Acta ; 1863(6 Pt A): 1082-92, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26876155

RESUMO

BACKGROUND: Oral mucositis (OM) is a relevant adverse effect of anticancer therapy involving ionizing radiation (IR) and doxorubicin (Doxo). Because DNA damage of keratinocytes is causative for the pathogenesis of OM, we aim to identify pharmacological measures for geno- and cytoprotection of keratinocytes. METHODS: We investigated the influence of the lipid-lowering drug lovastatin on cell death, proliferation and DNA damage response (DDR) mechanisms of human keratinocytes following treatment with IR and Doxo. RESULTS: Lovastatin protected keratinocytes from the cytotoxic and genotoxic effects of IR and Doxo as shown by a diminished induction of apoptosis as well as a reduced formation and slightly improved repair of DNA damage following Doxo and IR treatment, respectively. Lovastatin selectively blocked the activation of Chk1 and ATR kinases following treatment with IR, Doxo and the ribonucleotide reductase inhibitor hydroxyurea, indicating that the statin antagonizes ATR/Chk1-regulated replicative stress responses. Part of the cytoprotective activity of lovastatin seems to rest on a delayed entry of lovastatin treated cells into S-phase. Yet, because the statin also protected non-proliferating keratinocytes from IR- and Doxo-induced cytotoxicity, cell cycle independent protective mechanisms are involved, too. CONCLUSIONS: Lovastatin attenuates pro-toxic DNA damage-related responses of keratinocytes stimulated by OM-inducing anticancer therapeutics. The data encourage forthcoming in vivo and clinical studies addressing the usefulness of statins in the prevention of OM.


Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Queratinócitos/efeitos dos fármacos , Lovastatina/farmacologia , Adulto , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Quinase 1 do Ponto de Checagem , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Histonas/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Proteínas Quinases/metabolismo , Radiação Ionizante , Fatores de Tempo
19.
Toxicol Appl Pharmacol ; 292: 103-14, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26739623

RESUMO

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Lovastatina/farmacologia , Animais , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cisplatino/antagonistas & inibidores , Dano ao DNA/fisiologia , Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ratos
20.
J Exp Clin Cancer Res ; 34: 144, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26606927

RESUMO

BACKGROUND: Tumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro. METHODS: Magnetic- and fluorescence-activated- cell sorting were used for separation of CD90(+) and CD90(-) UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs. RESULTS: We observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90(+) cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90(+) cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90(+) cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes. CONCLUSIONS: Our findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Queratina-14/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Antígenos Thy-1/metabolismo , Neoplasias Uretrais/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunofenotipagem , Gradação de Tumores , Células-Tronco Neoplásicas/patologia , Via de Sinalização Wnt/efeitos dos fármacos
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