RESUMO
Neuroblastoma (NB) is a pediatric solid cancer with high fatality, relapses, and acquired resistance to chemotherapy, that requires new therapeutic approaches to improve survival. LGR5 is a receptor that potentiates WNT/signaling pathway and has been reported to promote development and survival in several adult cancers. In this study we investigated LGR5 expression in a panel of NB cell lines with acquired resistance to vincristine or doxorubicin. We show LGR5-LRP6 cooperation with enhanced expression in drug resistant NB cell lines compared to parental cells, suggesting a role for LGR5 in the emergence of drug resistance, warranting further investigation.
Assuntos
Neuroblastoma , Via de Sinalização Wnt , Criança , Humanos , Proteínas Wnt/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/uso terapêuticoRESUMO
During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.
Assuntos
Apoptose/fisiologia , Dano ao DNA , Instabilidade Genômica , Membranas Mitocondriais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p19/deficiência , Inibidor de Quinase Dependente de Ciclina p19/genética , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Nitrofenóis/farmacologia , Permeabilidade , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
AIM: A UK programme, led by the National Institute for Health Research (NIHR) ( https://www.nihr.ac.uk ) and coordinated by Applied Research Collaborations (ARC), ( https://www.nihr.ac.uk/explore-nihr/support/collaborating-in-applied-health-research.htm ) aimed to identify and select evidence-based, implementation-ready service innovations for evaluation. The programme focused on seven areas of health provision. We report on a prioritisation process designed to identify and assess innovations in one of these areas: child and maternal health (CH&M). METHODS: We developed a three-stage, online, stakeholder driven process to 1) identify, 2) assess and prioritise and 3) select evidence-based interventions or service models, using crowdsourcing to identify projects and the APEASE criteria to assess and select projects. A brief evidence review was conducted for all initial suggestions to identify those with the largest evidence-base to take forward for ranking by stakeholders. Stakeholder workshops considered and ranked these suggestions using the APEASE criteria. We then conducted in-depth evidence reviews for the highest ranked suggestions. The Project Management Group and Advisory Board used these reviews and the APEASE criteria to select the final projects. RESULTS: We received 32 initial suggestions from a range of clinicians, practitioners and researchers. Fourteen of the most evidence-based suggestions were considered and ranked at four themed stakeholder workshops. Nine suggestions were ranked for further in-depth evidence review and a final four projects were selected for implementation evaluation using the APEASE criteria. These were: 1. Maternal Mental Health Services Multidisciplinary Teams 2. Early years tooth brushing programme 3. Trauma-focused CBT for young people in care and 4. Independent Domestic Violence Advisors in maternity settings. Feedback from participants suggested that having public representatives participating in all stakeholder meetings, rather than being consulted separately, focused discussions clearly on patient benefit rather than research aims. CONCLUSIONS: The stakeholder-driven process achieved its aim of identifying, prioritising and assessing and selecting, evidence-based projects for wider implementation and evaluation. The concurrent process could be adapted by other researchers or policy makers.
Assuntos
Saúde da Criança , Pesquisadores , Adolescente , Criança , Medicina Baseada em Evidências , Feminino , Humanos , GravidezRESUMO
OBJECTIVE: To assess knowledge and practices related to dengue management among physicians. METHODS: The cross-sectional study was conducted at hospitals in Islamabad, Lahore, Faisalabad, Peshawar, Quetta and Karachi between June and December 2012Physicians from public and private sectors filled a self-administered questionnaire about dengue knowledge and its management practices. A maximum score of 100 was assigned to the knowledge portion. Data was analysed using SPSS 15. RESULTS: A total of 400 subjects participated in the study; 200(50%) each from public and private hospitals. Of them, 223(56%) were males; 268(67%) were in the 21-30 years age bracket. The highest score was recorded in Quetta 67 followed by 65 in Karachi, 62 in Lahore, Faisalabad, Peshawar and 59 in Islamabad. Of the total, 200 (50%) were not aware that leucopenia is a criterion for diagnosing probable dengue. Similarly 140 (35%) did not know the criteria for diagnosing dengue haemorrhagic fever and warning signs of severe dengue. Total of 204 (51%) were not aware of the criteria for discharging of the admitted cases. There was no significant difference between dengue knowledge of the physicians belonging to public and private sectors (p>0.05). CONCLUSIONS: Quite a large number of physicians lacked knowledge of probable diagnosis of dengue and appropriate time to discharge the patients.
Assuntos
Competência Clínica/normas , Dengue , Médicos Hospitalares , Dengue Grave , Adulto , Estudos Transversais , Dengue/diagnóstico , Dengue/terapia , Gerenciamento Clínico , Feminino , Pesquisas sobre Atenção à Saúde , Conhecimentos, Atitudes e Prática em Saúde , Médicos Hospitalares/normas , Médicos Hospitalares/estatística & dados numéricos , Humanos , Contagem de Leucócitos/métodos , Masculino , Avaliação das Necessidades , Paquistão , Alta do Paciente/normas , Setor Privado/estatística & dados numéricos , Setor Público/estatística & dados numéricos , Dengue Grave/diagnóstico , Dengue Grave/terapia , Inquéritos e QuestionáriosRESUMO
Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with "replication factories" and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro These data identify RS as a cancer stem cell-specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060-71. ©2018 AACR.
Assuntos
Carcinogênese , Glioblastoma/genética , Recidiva Local de Neoplasia/genética , Células-Tronco Neoplásicas , Tolerância a Radiação/genética , Antígeno AC133/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
From a set of weakly potent lead compounds, using in silico screening and small library synthesis, a series of 2-alkyl-3-aryl-3-alkoxyisoindolinones has been identified as inhibitors of the MDM2-p53 interaction. Two of the most potent compounds, 2-benzyl-3-(4-chlorophenyl)-3-(3-hydroxypropoxy)-2,3-dihydroisoindol-1-one (76; IC(50) = 15.9 +/- 0.8 microM) and 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (79; IC(50) = 5.3 +/- 0.9 microM), induced p53-dependent gene transcription, in a dose-dependent manner, in the MDM2 amplified, SJSA human sarcoma cell line.
Assuntos
Antineoplásicos/síntese química , Indóis/síntese química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.
Assuntos
Glioma/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Fosfatase 2/metabolismo , Animais , Glioma/patologia , Humanos , Camundongos , TransfecçãoRESUMO
Glioblastoma is the most common form of primary brain tumor in adults and is essentially incurable. Despite aggressive treatment regimens centered on radiotherapy, tumor recurrence is inevitable and is thought to be driven by glioblastoma stem-like cells (GSC) that are highly radioresistant. DNA damage response pathways are key determinants of radiosensitivity but the extent to which these overlapping and parallel signaling components contribute to GSC radioresistance is unclear. Using a panel of primary patient-derived glioblastoma cell lines, we confirmed by clonogenic survival assays that GSCs were significantly more radioresistant than paired tumor bulk populations. DNA damage response targets ATM, ATR, CHK1, and PARP1 were upregulated in GSCs, and CHK1 was preferentially activated following irradiation. Consequently, GSCs exhibit rapid G2-M cell-cycle checkpoint activation and enhanced DNA repair. Inhibition of CHK1 or ATR successfully abrogated G2-M checkpoint function, leading to increased mitotic catastrophe and a modest increase in radiation sensitivity. Inhibition of ATM had dual effects on cell-cycle checkpoint regulation and DNA repair that were associated with greater radiosensitizing effects on GSCs than inhibition of CHK1, ATR, or PARP alone. Combined inhibition of PARP and ATR resulted in a profound radiosensitization of GSCs, which was of greater magnitude than in bulk populations and also exceeded the effect of ATM inhibition. These data demonstrate that multiple, parallel DNA damage signaling pathways contribute to GSC radioresistance and that combined inhibition of cell-cycle checkpoint and DNA repair targets provides the most effective means to overcome radioresistance of GSC.
Assuntos
Dano ao DNA , Glioblastoma/genética , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Tolerância a Radiação/genética , Transdução de Sinais/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quinase 1 do Ponto de Checagem , Reparo do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Humanos , Mitose/efeitos dos fármacos , Mitose/genética , Mitose/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacosRESUMO
Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU-55933. We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC's display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation. Inhibition of ATM kinase by KU-55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6-3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU-55933 in GBM CSCs. ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Glioblastoma/radioterapia , Morfolinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Células-Tronco Neoplásicas/enzimologia , Pironas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Raios gama , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos da radiação , Masculino , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Tolerância a Radiação/efeitos da radiação , Células Tumorais CultivadasRESUMO
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.
Assuntos
Neoplasias Encefálicas/metabolismo , Proliferação de Células , Glioblastoma/metabolismo , Glutamato-Amônia Ligase/metabolismo , Glutamina/metabolismo , Nucleotídeos/biossíntese , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Células Cultivadas , Ciclo do Ácido Cítrico , Técnicas de Cocultura , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Glutamato-Amônia Ligase/genética , Ácido Glutâmico/metabolismo , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
OBJECTIVE: An analysis of a 5-year clinical experience in the management of gestational trophoblastic tumours in a tertiary care hospital. DESIGN: A prospective cohort follow-up study. PLACE AND DURATION OF STUDY: The study was conducted at Hayatabad Medical Complex, Peshawar from 1998 to 2003. PATIENTS AND METHODS: A total of 30 cases were managed and a detailed analysis of these patients was done. Of these 13 followed Hydatidiform Mole, 10 after abortion and 7 after a term pregnancy. RESULTS: Out of 30 cases of gestational trophoblastic tumour, 63.3% were between 21 and 38 years of age. Ninety percent of the patients presented with vaginal bleeding, while life-threatening hemorrhage occurred in 23.3%of the cases.43.3% of the patients had hydatidiform mole as an antecedent pregnancy and 36.7% of the patients presented within four months of the antecedent pregnancy. Blood groups O and B were most frequently encountered i.e. in 40% and 33.3% of the cases. Metastatic disease was present in 46.6% of the cases, of which 8 were high risk and one was of medium risk group. Major sites of metastasis were lungs (33.3%) and vagina (30%). Serum BHCG of 40,000 miu / ml and above was present in 53.3% of the cases (P=0.016) and number of metastasis >8 were found in 16.7% cases (P=0.001). Prior chemotherapy was given in only 2 patients and both of them died due to resistance. Chemotherapy was given to 100% of patients; survival was 100% in low-risk group and 50% in high-risk group (P=0.004). Overall mortality was 20% i.e. 6 patients died of the disease. Major side effects of chemotherapy were stomatitis (66.6%), alopecia (56.6%), low hemoglobin (60%), weight loss and recurrent infection. CONCLUSION: Late diagnosis, previously failed chemotherapy and high WHO prognostic scores are major risk factors affecting outcome in these patients. Hence every female in reproductive age group with unexplained bleeding per vaginum should be investigated with serum BHCG (Beta human chorionic gonadotrophin).
Assuntos
Doença Trofoblástica Gestacional/diagnóstico , Doença Trofoblástica Gestacional/terapia , Adulto , Feminino , Seguimentos , Humanos , Paquistão , Estudos Prospectivos , Resultado do TratamentoRESUMO
BACKGROUND: A safe motherhood initiative is a global effort to reduce maternal mortality and morbidity. This aims to ensure improvement in the quality and safety of lives of women through adoption of health and non-health strategies. Antenatal care is a branch of preventive medicine dealing with prevention and early detection of pregnancy disorders. It is the key to modern obstetrics. METHODS: It was a descriptive type of study to find out the attitude of the women towards utilization of antenatal care facility at a newly commissioned hospital, Hayatabad Medical Complex, Peshawar, Pakistan in the first year of its working. RESULTS: We recorded 980 patients. All of them were married. Monthly attendance showed increasing trend. In this study 72.44% of the women were of 25-35 year of age while 11.42% were teenager. Multiparity was recorded in 38.97% and 12.14% had more then nine pregnancies. Six hundred and fifty patients were living near the hospital. 2.24% patients visited more than six times and 63.26% had one visit only, with the rest visiting for 2-5 times. Thirty patients had medical disorders. Obstetrical diseases were detected in 194 patients. Pregnancy losses contributed to 146 women and 3 women had more then 6 losses. 254 women selected hospital delivery. Out of then 160 women were booked in the 3rd trimester. Caesarean section rate among those women was 16.4%. CONCLUSIONS: The conclusion drawn was that the women living near the hospital used the facility. Antenatal care should be provided to the women at the doorstep of their house. There is a system of lady health workers that should be expanded to cover all areas. Basic health units must be fully equipped and staffed. Communication system should be improved. There is a need to include health education in the curricula of primary education.
Assuntos
Atitude Frente a Saúde , Parto Obstétrico/estatística & dados numéricos , Serviços de Saúde Materna/estatística & dados numéricos , Mulheres/psicologia , Feminino , Humanos , Paquistão , GravidezRESUMO
OBJECTIVE: To assess the quality and patient satisfaction in Endoscopy Unit of Shifa International Hospital. STUDY DESIGN: Cross-sectional survey. PLACE AND DURATION OF STUDY: Division of Gastroenterology, Shifa International Hospital, Islamabad, Pakistan, from July 2011 to January 2012. METHODOLOGY: Quality and patient satisfaction after the endoscopic procedure was assessed using a modified GHAA-9 questionnaire. Data was analyzed using SPSS version 16. RESULTS: A total of 1028 patients were included with a mean age of 45 ± 14.21 years. Out of all the procedures, 670 (65.17%) were gastroscopies, 181 (17.60%) were flexible sigmoidoscopies and 177 (17.21%) were colonoscopies. The maximum unsatisfactory responses were on the waiting time before the procedure (13.13 %), followed by unsatisfactory explanation of the procedure and answers to questions (7.58%). Overall, unsatisfied impression was 4.86%. The problem rate was 6.22%. CONCLUSION: The quality of procedures and level of satisfaction of patients undergoing a gastroscopy or colonoscopy was generally good. The factors that influence the satisfaction of these patients are related to communication between doctor and patient, doctor's manner and waiting time for the procedure. Feedback information in an endoscopy unit may be useful in improving standards, including the performance of endoscopists.
Assuntos
Endoscopia Gastrointestinal/normas , Satisfação do Paciente/estatística & dados numéricos , Garantia da Qualidade dos Cuidados de Saúde , Indicadores de Qualidade em Assistência à Saúde , Adulto , Idoso , Atitude do Pessoal de Saúde , Comunicação , Estudos Transversais , Feminino , Pesquisas sobre Atenção à Saúde , Hospitais Universitários , Humanos , Masculino , Pessoa de Meia-Idade , Paquistão , Relações Médico-Paciente , Inquéritos e Questionários , Fatores de TempoRESUMO
Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile as an MDM2-p53 inhibitor (IC50 = 12.3 µM). MDM2-p53 and MDMX-p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC50 = 0.11 µM; MDMX IC50 = 4.2 µM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (MDM2 IC50 = 0.15 µM; MDMX IC50 = 4.2 µM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX-p53 activity but not for MDM2-p53 inhibition.
RESUMO
SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-Δ2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-Δ2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-Δ2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-Δ2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-Δ2/9 binds p53 protein. SIRT1-Δ2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-Δ2/9 prior to damage. In turn, basal p53 downregulates SIRT1-Δ2/9 RNA levels, while stress-activated p53 eliminates SIRT1-Δ2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-Δ2/9 in a range of human cancers. Exogenous SIRT1-Δ2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-Δ2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Sirtuína 1/genética , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Linhagem Celular Tumoral , Dano ao DNA , Deleção de Genes , Humanos , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND AND OBJECTIVE: The purpose of the study was to evaluate the perception of influence of music among surgeons, anesthesiologist and nurses in our hospital as well as to critically evaluate whether music can be used as an aid in improving the work efficiency of medical personnel in the operation theatre (OT). MATERIALS AND METHODS: A prospective, questionnaire-based cross-sectional study was conducted. A total of 100 randomly selected subjects were interviewed, which included 44 surgeons, 25 anesthesiologists and 31 nurses. Statistical package for social sciences (SPSS) Windows Version 16 software was used for statistical evaluation. RESULTS: Most of the OT medical personnel were found to be aware of the beneficial effects of music, with 87% consenting to the playing of music in the OT. It was also found that most participants agreed to have heard music on a regular basis in the OT, while 17% had heard it whenever they have been to the OT. CONCLUSIONS: Majority of the respondent's preferred playing music in the OT which helped them relax. It improved the cognitive function of the listeners and created a sense of well being among the people and elevated mood in them. Music helped in reducing the autonomic reactivity of theatre personnel in stressful surgeries allowing them to approach their surgeries in a more thoughtful and relaxed manner. Qualitative, objective and comprehensive effect of specific music types varied with different individuals. Music can aid in improving the work efficiency of medical personnel in the OT. The study has reinforced the beneficial effects of playing music in the OT outweighing its deleterious outcomes.
RESUMO
Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 µM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 µM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.
Assuntos
Antineoplásicos/síntese química , Isoindóis/síntese química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoindóis/química , Isoindóis/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation. CONCLUSIONS/SIGNIFICANCE: We characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline.