RESUMO
Background: Patient-reported outcomes (pros) are essential to capture the patient's perspective and to influence care. Although pros and pro measures are known to have many important benefits, they are not consistently being used and there is there no Canadian pros oversight. The Position Statement presented here is the first step toward supporting the implementation of pros in the Canadian health care setting. Methods: The Canadian pros National Steering Committee drafted position statements, which were submitted for stakeholder feedback before, during, and after the first National Canadian Patient Reported Outcomes (canpros) scientific conference, 14-15 November 2019 in Calgary, Alberta. In addition to the stakeholder feedback cycle, a patient advocate group submitted a section to capture the patient voice. Results: The canpros Position Statement is an outcome of the 2019 canpros scientific conference, with an oncology focus. The Position Statement is categorized into 6 sections covering 4 theme areas: Patient and Families, Health Policy, Clinical Implementation, and Research. The patient voice perfectly mirrors the recommendations that the experts reached by consensus and provides an overriding impetus for the use of pros in health care. Conclusions: Although our vision of pros transforming the health care system to be more patient-centred is still aspirational, the Position Statement presented here takes a first step toward providing recommendations in key areas to align Canadian efforts. The Position Statement is directed toward a health policy audience; future iterations will target other audiences, including researchers, clinicians, and patients. Our intent is that future versions will broaden the focus to include chronic diseases beyond cancer.
Assuntos
Atenção à Saúde/estatística & dados numéricos , Oncologia/estatística & dados numéricos , Neoplasias/terapia , Medidas de Resultados Relatados pelo Paciente , Assistência Centrada no Paciente/estatística & dados numéricos , Canadá , Atenção à Saúde/métodos , Atenção à Saúde/normas , Humanos , Oncologia/métodos , Oncologia/normas , Neoplasias/diagnóstico , Assistência Centrada no Paciente/métodos , Assistência Centrada no Paciente/normas , Qualidade de VidaRESUMO
A nine-year-old male, neutered, pug was presented for investigation of progressive ambulatory paraparesis and pelvic limb ataxia of three months' duration. Magnetic resonance imaging was suggestive of caudal thoracic syringohydromyelia with an adjacent intradural arachnoid cyst. The cyst was marsupialised following dorsal laminectomy. Neurological status had improved 10 weeks following surgery when repeat magnetic resonance imaging revealed reduced spinal cord compression both as a result of resolution of the cyst and reduction in size of the syringohydromyelia. At 17 months following surgery, the dog showed further improvements in neurological status, exhibiting mild pelvic limb ataxia and proprioceptive deficits. Improved cerebrospinal fluid flow following surgery may have played a role in the improvement in both conditions. The presence of syringohydromyelia in this context does not preclude a favourable clinical outcome following surgical management.
Assuntos
Cistos Aracnóideos/veterinária , Doenças do Cão/cirurgia , Laminectomia/veterinária , Siringomielia/veterinária , Animais , Cistos Aracnóideos/cirurgia , Cães , Imageamento por Ressonância Magnética/veterinária , Masculino , Siringomielia/cirurgia , Resultado do TratamentoRESUMO
Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER)-positive patients. We have previously reported that hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) inhibits ERα activity by competing with ERα for binding to cyclin T1, a subunit of positive transcription elongation b (P-TEFb). This results in the inhibition of the phosphorylation of RNA polymerase II (RNAPII) at serine 2 and the inhibition of transcription elongation of ERα target genes. As HEXIM1 can inhibit ER activity, we examined whether it has a critical role in the inhibitory effects of tamoxifen on ER. We observed that tamoxifen-induced HEXIM1 recruitment to the promoter region of ER target genes and decreased the recruitment of cyclin T1 and serine 2 phosphorylated RNAPII to the coding regions of these genes. Conversely, in cells wherein HEXIM1 expression has been downregulated we observed attenuation of the inhibitory effects of tamoxifen on estrogen-induced cyclin T1 recruitment to coding regions of ER target genes. As a consequence, downregulation of HEXIM1 resulted in the attenuation of the repressive effects of tamoxifen on estrogen-induced gene expression and proliferation. Conferring clinical relevance to our studies is our analysis of human breast cancer tissue samples that indicated association of lower expression of HEXIM1 with tumor recurrence in patients who received tamoxifen. Our studies provide a better understanding of the mechanistic basis for the inhibitory effect of tamoxifen on ER activity and may suggest new therapeutic targets for the treatment of tamoxifen-resistant breast cancer.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/farmacologia , Proteínas de Ligação a RNA/fisiologia , Tamoxifeno/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina T/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/fisiologia , Feminino , Humanos , Recidiva Local de Neoplasia/prevenção & controle , Fosforilação , RNA Polimerase II/metabolismo , Fatores de Transcrição , Fatores de Elongação da Transcrição/metabolismoRESUMO
Five cats were treated for a fracture of the medial malleolus, 10 for a fracture of the lateral malleolus and 15 for fractures of both malleoli. Open reduction and internal fixation with Kirschner wires (K-wires) with or without a tension band wire was applied to 26 of the fractures. Unilateral-uniplanar or bilateral-uniplanar transarticular external skeletal fixators were applied to provide coaptation in 19 cases and appeared to be well tolerated. In 24 cases fracture healing was assessed radiographically between four and eight weeks after treatment. In seven cases fracture healing was good, in 12 cases it was reasonable, in four cases it was poor and in one case with two fractures healing was poor in one and reasonable in the other. The 12 fractures treated by open reduction and internal fixation with K-wires and a tension band wire all showed evidence of good or reasonable healing. The nine fractures treated by open reduction and internal fixation with K-wires without a tension band wire showed evidence of good or reasonable healing in six cases and poor healing in three cases. Complications occurred in nine of the cats and included persistent talocrural instability, soft tissue necrosis, implant migration and external fixator pin breakage. Twenty-one of the cats were followed up by telephone questionnaire for between 5.5 and 84 months; the owner satisfaction was excellent in 17 cases, good in two cases, moderate in one case and poor in one case.
Assuntos
Gatos/lesões , Gatos/cirurgia , Fíbula/lesões , Fraturas Ósseas/veterinária , Procedimentos Ortopédicos/veterinária , Animais , Fios Ortopédicos/veterinária , Fixadores Externos/veterinária , Feminino , Seguimentos , Consolidação da Fratura , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/cirurgia , Masculino , Procedimentos Ortopédicos/métodos , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/veterinária , Radiografia , Inquéritos e Questionários , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/cirurgia , Fraturas da Tíbia/veterinária , Resultado do TratamentoRESUMO
Twenty-four simple or comminuted supracondylar and diaphyseal femoral fractures in cats, which had been treated by retrograde insertion of a new 3.5 mm titanium interlocking nail (IN) from the intercondylar notch, were evaluated between June 2000 and October 2004 at the Ecole Nationale Vétérinaire d'Alfort. Patient data (species, breed, weight, and age), fracture characteristics, details of the surgery, post-operative complications and radiographic follow-up were recorded. The mean body weight was 3.75 kg and the cats ranged in age between four and 66 months (mean 29 months). The IN had a diameter of 3.5 mm and a length of 100, 109 or 119 mm, and were all fixed in a static position (two screws in seven cats, three screws in 10 cats and four screws in seven cats). Cerclage wires were used in seven cats and an autogenous bone graft was used in two cats. Screw or nail breakage were not recorded. Nineteen fractures healed without any complications, three cats died during the post-operative period from unknown causes, and two cats showed delayed bone healing. Twenty cats were considered to have an excellent limb function at one month. One cat with a sciatic injury was non-weight bearing for several months. Radiographic signs of degenerative joint disease of the stifle joint were not observed except in the cat with the sciatic nerve injury. The results of this study suggest that this new 3.5 mm titanium IN can be introduced from the intercondylar notch and be used in static fixation mode to stabilize supracondylar and diaphyseal femoral fractures in cats.
Assuntos
Pinos Ortopédicos/veterinária , Gatos/cirurgia , Fraturas do Fêmur/veterinária , Animais , Gatos/lesões , Diáfises/lesões , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/epidemiologia , Fraturas do Fêmur/cirurgia , Irlanda/epidemiologia , Complicações Pós-Operatórias/veterinária , Radiografia , Registros/veterinária , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVES: To document the histories, clinical findings, and management of seven puppies with laryngeal collapse occurring secondarily to brachycephalic airway syndrome. METHODS: Seven brachycephalic puppies aged between 4.5 and six months underwent surgery for management of brachycephalic airway syndrome following presentation for exercise intolerance and increased respiratory noise and effort. RESULTS: Stenotic nares of varying severity and an elongated soft palate were common to all dogs. All dogs had tracheal hypoplasia and this was severe in four dogs. Laryngeal collapse was present in all dogs. Two dogs had stage I, four dogs stage II, and one dog stage III laryngeal collapse. The dog with stage III laryngeal collapse and one dog with stage II laryngeal collapse died. There was no apparent association between the changes evident on thoracic radiographs or the degree of tracheal hypoplasia and postoperative outcome. CLINICAL SIGNIFICANCE: The development of severe secondary laryngeal changes in dogs aged six months or less supports the suggestion that immature brachycephalic dogs should undergo assessment and, if indicated, surgery as soon as any clinical signs of BAS are apparent.
Assuntos
Obstrução das Vias Respiratórias/veterinária , Doenças do Cão/diagnóstico , Doenças da Laringe/veterinária , Complicações Pós-Operatórias/veterinária , Obstrução das Vias Respiratórias/genética , Obstrução das Vias Respiratórias/cirurgia , Animais , Cruzamento , Constrição Patológica/genética , Constrição Patológica/cirurgia , Constrição Patológica/veterinária , Doenças do Cão/genética , Doenças do Cão/cirurgia , Cães , Doenças da Laringe/diagnóstico , Doenças da Laringe/genética , Doenças da Laringe/cirurgia , Cavidade Nasal/anormalidades , Cavidade Nasal/cirurgia , Palato Mole/anormalidades , Palato Mole/cirurgia , Complicações Pós-Operatórias/diagnóstico , Estudos RetrospectivosAssuntos
Apendicite/etiologia , Colonoscopia/efeitos adversos , Insuficiência de Múltiplos Órgãos/etiologia , Idoso , Apendicite/diagnóstico , Apendicite/cirurgia , Evolução Fatal , Humanos , Laparotomia , Masculino , Insuficiência de Múltiplos Órgãos/diagnóstico , Insuficiência de Múltiplos Órgãos/cirurgiaRESUMO
beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Camptotecina/farmacologia , Caspases/metabolismo , Dicumarol/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Transformação Genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
beta-Lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Downstream signaling pathway(s) that initiate apoptosis following treatment with beta-Lap have not been elucidated. Since calpain activation was suspected in beta-Lap-mediated apoptosis, we examined alterations in Ca(2+) homeostasis using NQO1-expressing MCF-7 cells. beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+), from endoplasmic reticulum Ca(2+) stores, comparable to thapsigargin exposures. 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca(2+) chelator, blocked early increases in Ca(2+) levels and inhibited beta-Lap-mediated mitochondrial membrane depolarization, intracellular ATP depletion, specific and unique substrate proteolysis, and apoptosis. The extracellular Ca(2+) chelator, EGTA, inhibited later apoptotic end points (observed >8 h, e.g. substrate proteolysis and DNA fragmentation), suggesting that later execution events were triggered by Ca(2+) influxes from the extracellular milieu. Collectively, these data suggest a critical, but not sole, role for Ca(2+) in the NQO1-dependent cell death pathway initiated by beta-Lap. Use of beta-Lap to trigger an apparently novel, calpain-like-mediated apoptotic cell death could be useful for breast and prostate cancer therapy.
Assuntos
Cálcio/metabolismo , Morte Celular , Naftoquinonas/metabolismo , Transdução de Sinais , 4-Nitroquinolina-1-Óxido/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose , Western Blotting , Neoplasias da Mama/metabolismo , Divisão Celular , Quelantes/farmacologia , Citosol/metabolismo , Fragmentação do DNA , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/metabolismo , Feminino , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Potenciais da Membrana , Microscopia Confocal , Mitocôndrias/metabolismo , Modelos Biológicos , Naftoquinonas/farmacologia , Neoplasias da Próstata/metabolismo , Quinolonas/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Clusterin [CLU, a.k.a. TRPM-2, SGP-2, or ionizing radiation (IR)-induced protein-8 (XIP8)] was implicated in apoptosis, tissue injury, and aging. Its function remains elusive. We reisolated CLU/XIP8 by yeast two-hybrid analyses using as bait the DNA double-strand break repair protein Ku70. We show that a delayed (2-3 days), low-dose (0.02-10 Gy) IR-inducible nuclear CLU/XIP8 protein coimmunoprecipitated and colocalized (by confocal microscopy) in vivo with Ku70/Ku80, a DNA damage sensor and key double-strand break repair protein, in human MCF-7:WS8 breast cancer cells. Overexpression of nuclear CLU/XIP8 or its minimal Ku70 binding domain (120 aa of CLU/XIP8 C terminus) in nonirradiated MCF-7:WS8 cells dramatically reduced cell growth and colony-forming ability concomitant with increased G(1) cell cycle checkpoint arrest and increased cell death. Enhanced expression and accumulation of nuclear CLU/XIP8-Ku70/Ku80 complexes appears to be an important cell death signal after IR exposure.
Assuntos
Antígenos Nucleares , Morte Celular/fisiologia , DNA Helicases , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/fisiologia , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenocarcinoma/patologia , Motivos de Aminoácidos , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Clusterina , Dano ao DNA , DNA Complementar/genética , DNA de Neoplasias/efeitos da radiação , Proteína Quinase Ativada por DNA , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Biblioteca Gênica , Genes Reporter , Glicoproteínas/química , Glicoproteínas/genética , Proteínas de Fluorescência Verde , Humanos , Autoantígeno Ku , Proteínas Luminescentes/genética , Microscopia Confocal , Ligação Proteica , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/genética , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Cisteína Endopeptidases/metabolismo , Naftoquinonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Células Tumorais CultivadasRESUMO
beta-Lapachone activates a novel apoptotic response in a number of cell lines. We demonstrate that the enzyme NAD(P)H:quinone oxidoreductase (NQO1) substantially enhances the toxicity of beta-lapachone. NQO1 expression directly correlated with sensitivity to a 4-h pulse of beta-lapachone in a panel of breast cancer cell lines, and the NQO1 inhibitor, dicoumarol, significantly protected NQO1-expressing cells from all aspects of beta-lapachone toxicity. Stable transfection of the NQO1-deficient cell line, MDA-MB-468, with an NQO1 expression plasmid increased apoptotic responses and lethality after beta-lapachone exposure. Dicoumarol blocked both the apoptotic responses and lethality. Biochemical studies suggest that reduction of beta-lapachone by NQO1 leads to a futile cycling between the quinone and hydroquinone forms, with a concomitant loss of reduced NAD(P)H. In addition, the activation of a cysteine protease, which has characteristics consistent with the neutral calcium-dependent protease, calpain, is observed after beta-lapachone treatment. This is the first definitive elucidation of an intracellular target for beta-lapachone in tumor cells. NQO1 could be exploited for gene therapy, radiotherapy, and/or chemopreventive interventions, since the enzyme is elevated in a number of tumor types (i.e. breast and lung) and during neoplastic transformation.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Divisão Celular/efeitos dos fármacos , Naftoquinonas/farmacologia , Quinona Redutases/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Redutases do Citocromo/metabolismo , Citocromo-B(5) Redutase , Dicumarol/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Modelos Biológicos , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADPH-Ferri-Hemoproteína Redutase , Proteínas/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Vitamina K/farmacologiaRESUMO
The efficacy of topoisomerase (Topo) I-active drugs may be improved by better understanding the molecular and cellular responses of tumor compared to normal cells after genotoxic insults. Ionizing radiation (IR) + Topo I-active drugs (e.g., Topotecan) caused synergistic cell killing in various human cancer cells, even in cells from highly radioresistant tumors. Topo I poisons had to be added either during or immediately after IR. Synergy was caused by DNA lesion modification mechanisms as well as by concomitant stimulation of two pathways of cell death: necrosis (IR) + apoptosis (Topo I poisons). Cumulative data favor a mechanism of synergistic cell killing caused by altered DNA lesion modification and enhanced apoptosis. However, alterations in cell cycle regulation may also play a role in the synergy between these two agents in certain human cancers. We recently showed that NF-kappa B, a known anti-apoptotic factor, was activated in various cancer cells after poisoning Topo I using clinically active drugs. NF-kappa B activation was dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. Cytoplasmic signaling leading to NF-kappa B activation after Topo I poisons was diminished in cytoplasts (lacking nuclei) and in CEM/C2 cells that expressed a mutant Topo I protein that did not interact with Topo I-active drugs. NF-kappa B activation was intensified in S-phase and blocked by aphidicolin, suggesting that activation was a result of double-strand break formation due to Topo I poisoning and DNA replication. Dominant-negative I kappa B expression augmented Topo I poison-mediated apoptosis. Elucidation of molecular signal transduction pathways after Topo I drug-IR combinations may lead to improved radiotherapy by blocking anti-apoptotic NF-kappa B responses. Recent data also indicate that synergy caused by IR + Topo I poisons is different from radiosensitization by beta-lapachone (beta-lap), a "reported" Topo I and II-alpha poison in vitro. In fact, beta-lap does not kill cells by poisoning either Topo I or II-alpha in vivo. Instead, the compound is "activated" by an IR (damage)-inducible enzyme, NAD(P)H:quinone oxidoreductase (NQO1), a gene cloned as x-ray-inducible transcript #3, xip3. Unlike the lesion modification pathway induced by IR + Topo I drugs, beta-lap kills cells via NQO1 futile cycle metabolism. Downstream apoptosis caused by beta-lap appears to be noncaspase-mediated, involving calpain or a calpain-like protease. Thus, although Topo I poisons or beta-lap in combination with IR both synergistically kill cancer cells, the mechanisms are very different.
Assuntos
Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Radiossensibilizantes/farmacologia , Inibidores da Topoisomerase I , Animais , Bovinos , DNA Topoisomerases Tipo I/fisiologia , Humanos , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
These studies were carried out to examine the capacity of alpha-difluoromethylornithine (DFMO) to modulate cell proliferation and apoptosis in cells of squamous cell carcinomas (SCCs) of the head and neck. Exposure of cells to DFMO (5 mM for 48 h) depleted intracellular putrescine and spermidine levels (greater than 5-fold) and inhibited proliferation of the cells without manifestation of cytotoxicity as measured by a clonogenic assay. Exposure of the cells to DFMO did not influence the survival response after exposure to single-dose radiation between 0 and 10 Gy. Treatment of polyamine-depleted cells with 200 nM staurosporine amplified apoptosis 65% (1.65-fold) over that in controls, as determined by flow cytometry. The increased apoptosis after DFMO treatment was effectively inhibited by the addition of 1 mM putrescine or spermidine. Cleavage of poly(ADP-ribose) polymerase (PARP) illustrated that the staurosporine treatment induced apoptosis in the cells within 6 h. Analysis of PARP cleavage indicated that treatment with DFMO accelerated the kinetics of progression of apoptosis but did not influence the sensitivity of cells to 10 nM-1 microM staurosporine. These data suggest an involvement of endogenous polyamines in modulation of proliferation kinetics and apoptosis in human SCCs and suggest opportunities to explore new therapeutic strategies in head and neck cancer patients to be treated with radiation therapy.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Eflornitina/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Poliaminas/metabolismo , Apoptose/efeitos da radiação , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Cinética , Poli(ADP-Ribose) Polimerases/metabolismo , Estaurosporina/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
HeLa X human skin fibroblast hybrid cells have been developed into a model for radiation-induced neoplastic transformation of human cells. Previous studies indicate that the appearance of neoplastically transformed foci in this system is delayed for several population doublings after irradiation and appears to involve the loss of putative tumor suppressor loci on fibroblast chromosomes 11 and 14. We now show that after treatment with 7 Gy of X-rays, transformed foci initiation correlates with delayed apoptosis initiated in the progeny of the irradiated cells after 10-12 cell divisions and with reduced plating efficiency (delayed death). The cells develop classic apoptotic morphology, positive terminal deoxynucleotidyl transferase-mediated nick end labeling and phosphatidylserine (annexin V) staining, and cleavage of poly(ADP-ribose) polymerase. In addition, a delayed induction of the p53 protein and the proapoptotic Bax protein is evident over a week after radiation exposure. We propose that a delayed build-up of mitosis-dependent genomic DNA damage or a loss of genetic material over time (10-12 cell divisions postirradiation) has two relevant outcomes: (a) cell death due to the delayed induction of a p53-dependent apoptosis; and (b) neoplastic transformation of a minor subset of survivors that has lost fibroblast chromosomes 11 and 14 (tumor suppressor loci for this system) and has either evaded apoptosis or not acquired enough genetic damage to induce apoptosis. It is postulated that both phenomena result from X-ray-induced, translesion-mediated genomic instability.
Assuntos
Apoptose , Transformação Celular Neoplásica , Células Híbridas/patologia , Células Híbridas/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Fibroblastos/patologia , Células HeLa/patologia , Humanos , Fatores de TempoRESUMO
Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.
Assuntos
Apoptose/efeitos dos fármacos , Indóis/farmacologia , Doenças de Niemann-Pick/patologia , Fotoquimioterapia , Humanos , Marcação In Situ das Extremidades Cortadas , Fármacos Fotossensibilizantes/farmacologia , Esfingomielina Fosfodiesterase/farmacologiaRESUMO
We previously demonstrated that beta-lapachone (beta-lap) killed cancer cells solely by apoptosis. Beta-Lap induced apoptosis in HL-60 cells in a dose-dependent manner as measured by flow cytometry and DNA ladder formation. Cell cycle changes, such as accumulations in S and G2-phases, were not observed. Apoptosis was accompanied by activation of caspase 3 and concomitant cleavage of poly(ADP-ribose) polymerase (PARP) to an 89 kDa polypeptide. PARP cleavage was blocked by zDEVD-fmk or zVAD-fmk, caspase-specific cleavage site inhibitors. Retrovirally introduced bcl-2 prevented beta-lap-mediated caspase 3 activation and PARP cleavage and increased the viability of Bcl-2-expressing HL-60 cells compared to cells with vector alone. Various beta-lap-related analogs (e.g., dunnione and naphthoquinone derivatives) induced equivalent apoptosis in HL-60 cells, but no compound was more effective than beta-lap. These data provide further evidence that the primary mode of cell killing by beta-lap is by the initiation and execution of apoptosis in human cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Naftoquinonas/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Vetores Genéticos , Células HL-60/citologia , Células HL-60/enzimologia , Humanos , Naftoquinonas/farmacocinética , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.
Assuntos
Corpo Estriado/efeitos dos fármacos , AMP Cíclico/biossíntese , Agonistas de Aminoácidos Excitatórios/farmacologia , Neurônios/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/agonistas , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Autorradiografia , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Imunofluorescência , Ácido Glutâmico/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Hidrólise , Hibridização In Situ , Neurônios/metabolismo , Fosfatidilinositóis/metabolismo , Ratos , Resorcinóis/farmacologiaRESUMO
Beta-lapachone (beta-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 microM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4-8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G1-S border. Apoptosis activated by beta-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 microM beta-lap. Topo I and Topo IIalpha levels decreased at > 24 h. Logarithmic-phase MCF-7 cells were not affected by < or = 1 microM beta-lap. In contrast, dramatic and irreversible G2-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 microM CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 microM) resulted in only approximately 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to > 5 microM CPT arrested at key cell cycle checkpoints (i.e., G1, S, and G2-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decreases in bcl-2, Topo I, Topo IIalpha, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed. Beta-lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.