RESUMO
PURPOSE: The purpose of this study was to investigate the changes in higher-order aberrations (HOAs), coma, and spherical aberrations (SAs) on the anterior, posterior, and total corneal surfaces after pterygium excision. METHODS: In this single-center study, we examined 19 eyes of 15 patients who underwent pterygium excision at Yokohama Minami Kyosai Hospital between January 2017 and December 2017. We also evaluated 25 eyes of 25 age-matched patients with no history of ocular disease as the control group. Corneal topography, total HOAs, coma, and SAs in all regions at 4 and 6 mm diameters were evaluated using anterior segment optical coherence tomography (CASIA SS-1000, Tomey, Japan). The pterygium area and extent were also assessed. RESULTS: Significant improvements in the HOAs, coma, and SAs at both diameters were observed in the total and anterior corneas from the first postoperative month. Notably, the posterior cornea showed significant improvements in HOAs (4 mm: P < 0.001 [log HOAs]; 6 mm: P = 0.001 [log HOAs]) and coma (4 mm: P = 0.003 [log coma], 6 mm: P = 0.002 [log coma]) within both diameters at 1 month postoperatively. A strong correlation was identified among the pterygium area, posterior HOAs, and coma (Spearman correlation = 0.651). Pterygium induced 2 D of astigmatism when extension exceeded 2.1 mm. CONCLUSIONS: HOAs in both the anterior and posterior corneas improved after pterygium excision. This finding underscores the importance of considering corneal aberrations on both anterior and posterior surfaces in pterygium management.
RESUMO
Tensional homeostasis is crucial for organ and tissue development, including the establishment of morphological and functional properties. Skin plays essential roles in waterproofing, cushioning and protecting deeper tissues by forming internal tension-distribution patterns, which involves aligning various cells, appendages and extracellular matrices (ECMs). The balance of traction force is thought to contribute to the formation of strong and pliable physical structures that maintain their integrity and flexibility. Here, by using a human skin equivalent (HSE), the horizontal tension-force balance of the dermal layer was found to clearly improve HSE characteristics, such as the physical relationship between cells and the ECM. The tension also promoted skin homeostasis through the activation of mechano-sensitive molecules such as ROCK and MRTF-A, and these results compared favourably to what was observed in tension-released models. Tension-induced HSE will contribute to analyze skin physiological functions regulated by tensional homeostasis as an alternative animal model.
Assuntos
Fenômenos Fisiológicos da Pele , Pele/citologia , Pele/efeitos dos fármacos , Amidas/farmacologia , Animais , Fenômenos Biomecânicos , Adesão Celular , Epiderme/fisiologia , Matriz Extracelular/fisiologia , Feminino , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Homeostase , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Piridinas/farmacologia , Pele/química , Estresse Mecânico , Técnicas de Cultura de TecidosRESUMO
The Japanese government suspended proactive recommendation of human papillomavirus (HPV) vaccination due to several reports of adverse events related to it in 2013. After that, the immunization rate of HPV vaccine quickly declined in Japan. Health science teachers (HSTs) are qualified and licensed teachers in charge of health care and health education for students in Japanese schools. HSTs have not recommended HPV vaccination to female students, since active governmental recommendation for HPV vaccination was halted for 5 y. We conducted a primary survey targeting HSTs (N = 39) and university students taking the HST training course (N = 123). In each group, awareness regarding HPV vaccine and disease burden was evaluated and factors related to and barriers to HPV vaccine recommendation were identified. The primary survey found that many HSTs and university students recognized their insufficient knowledge regarding the HPV vaccine. Based on the primary survey's results, infectious disease specialists, collaborating with Japanese HSTs, developed educational slide sets on HPV vaccine. A secondary survey was conducted before and after the lecture with HSTs (N = 162), where we evaluated their intelligibility and intention to recommend HPV vaccination for female students. In the post-lecture, secondary survey, the number of HSTs who recommended the HPV vaccine to their students had statistically increased from 76 before the lecture, to 103 (p < .05). An educational lecture using appropriate materials improved HSTs' vaccine confidence and intention to recommend the HPV vaccine to their students, verifying the study's hypothesis.
Assuntos
Infecções por Papillomavirus , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Adolescente , Feminino , Educação em Saúde , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Intenção , Japão , Infecções por Papillomavirus/prevenção & controle , Estudantes , Inquéritos e Questionários , VacinaçãoRESUMO
Graft insertion into the anterior chamber is one of the most important procedures for successful Descemet membrane endothelial keratoplasty (DMEK). Especially in eyes with fragile zonular fibers and a shallow anterior chamber, smooth graft insertion tends to become more difficult. Ophthalmic viscoelastic devices (OVDs) can usually help to retain the space in the anterior chamber and to improve the safety of manipulations during various ophthalmic surgeries. Therefore, we postulated that graft insertion into the anterior chamber could be improved by their use. The purpose of this study is to investigate the availability and efficacy of OVDs during graft insertion in DMEK surgery.A total of 11 eyes of 9 patients with bullous keratopathy who underwent DMEK were retrospectively analyzed. The cause of bullous keratopathy was corneal endothelial decompensation following laser iridotomy in all eyes. We used low viscous dispersive OVD (Opegan) to maintain the anterior chamber depth during graft insertion in all of the eyes.The graft insertion was uneventful in all of the eyes. The inserted graft was attached to the back surface of the cornea. However, 2 eyes needed rebubbling, and after rebubbling, all of the 2 grafts completely attached to the back surface of the cornea. The best spectacle-corrected visual acuity significantly improved 6 months after surgery (Pâ<â.001) and the central corneal thickness significantly decreased (Pâ<â.001).The use of OVD facilitates safer graft insertion during DMEK, and subsequently prevents endothelial cell loss, which leads to a successful procedure.
Assuntos
Perda de Células Endoteliais da Córnea/cirurgia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Terapia a Laser/efeitos adversos , Substâncias Viscoelásticas/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Câmara Anterior/cirurgia , Perda de Células Endoteliais da Córnea/etiologia , Lâmina Limitante Posterior/cirurgia , Endotélio Corneano/patologia , Endotélio Corneano/transplante , Feminino , Humanos , Masculino , Estudos Retrospectivos , Acuidade VisualRESUMO
PURPOSE: To describe a simple technique that uses posterior chandelier illumination during Descemet membrane endothelial keratoplasty in cases of severe bullous keratopathy (BK). METHODS: Five eyes of 4 patients with advanced BK undergoing Descemet membrane endothelial keratoplasty were retrospectively analyzed. The pupil of the host eye was not treated with mydriatic or miotic agents. The chandelier illuminator was inserted transconjunctivally into the vitreous cavity from the pars plana. RESULTS: In all eyes, BK was secondary to laser iridotomy, which was performed for prevention or treatment of angle closure glaucoma. The implanted graft was clearly confirmed in the anterior chamber using activated chandelier illumination. The graft was immediately attached to the host cornea, with eventual healing of BK in all eyes. No complication involving insertion or removal of the 25-gauge trocar and the chandelier illuminator was observed. No vision-threatening complication was observed in any of the 5 eyes. CONCLUSIONS: The chandelier illuminator provided good visibility of the anterior chamber and enhanced the safety of surgery by preventing formation of an inverted graft.
Assuntos
Doenças da Córnea/cirurgia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Iluminação/métodos , Idoso , Idoso de 80 Anos ou mais , Edema da Córnea/patologia , Lâmina Limitante Posterior/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Acuidade VisualRESUMO
BACKGROUND: The newly-synthesized naftopidil analog HUHS1015 suppresses tumor growth and induces apoptosis of cells from a variety of cancer types. The present study was conduced to assess the effect of HUHS1015 on human colonic cancer cells and to clarify the underlying mechanism. RESULTS: HUHS1015 reduced cell viability of Caco-2 and CW2 human colonic cancer cell lines in a concentration (0.3-100 mM)-dependent manner. HUHS1015 increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in both cell lines. In flow cytometry using propidium iodide and annexin V, HUHS1015 significantly increased the populations of cells undergoing primary necrosis, early apoptosis, and late apoptosis/secondary necrosis in both cell lines. In the cell-cycle analysis, HUHS1015 increased the proportion of the sub-G1 phase of cell, which corresponds to apoptotic cells. HUHS1015 perturbed the mitochondrial membrane potential and reduced the intracellular ATP level. HUHS1015 activated caspases 3, -4, -8, and -9, particularly caspase-3. HUHS1015 promoted cytochrome c release from the mitochondria. HUHS1015 significantly inhibited tumor growth in mice inoculated with CW2 cells. CONCLUSION: HUHS1015 induces necrosis by lowering the intracellular ATP level in association with mitochondrial damage and caspase-dependent apoptosis. This occurs in part by stimulating cytochrome c release from the mitochondria to activate caspase-9 followed by the effector caspase-3, responsible for suppression of colonic cancer proliferation in the mouse xenograft model.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Propanolaminas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Células CACO-2 , Caspases/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Necrose , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glycogen synthase kinase-3ß (GSK-3ß) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3ß by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3ß. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3ß than each independent treatment, to suppress amyloid ß (Aß)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas tau/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Células PC12 , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Ratos WistarRESUMO
BACKGROUND: The present study investigated the mechanism underlying the apoptosis of MKN28 human gastric cancer cells induced by the phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-D-PI) and its enantiomer diDCP-LA-L-PI. MATERIALS AND METHODS: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, enzymatic caspase assay, real-time reverse transcription-polymerase chain reaction (RT-PCR), and western blotting were carried-out. RESULTS: Both diDCP-LA-D-PI and diDCP-LA-L-PI induced caspase-independent apoptosis of MKN28 cells, with the potential for diDCP-LA-L-PI being much greater than that of diDCP-LA-D-PI. diDCP-LA-D-PI and diDCP-LA-L-PI accumulated apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) in the nucleus. CONCLUSION: diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 cells by accumulating AIF and AMID in the nucleus, with different potentials.
Assuntos
Fator de Indução de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfatidilinositóis/metabolismo , Neoplasias Gástricas/genética , Apoptose , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Estrutura Molecular , Neoplasias Gástricas/metabolismoRESUMO
1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Mesotelioma/patologia , Fosfatidiletanolaminas/farmacologia , Neoplasias Pleurais/patologia , Tiorredoxinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Humanos , Mesotelioma Maligno , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Amyloid-ß peptide 1-42 (Aß1-42) reduced PC-12 cell viability in a concentration (1-10 µM)- and treatment time (48-72 h)-dependent manner. Nicotine prevented Aß1-42-induced PC-12 cell death, but conversely, the α7 ACh receptor antagonist α-bungarotoxin enhanced Aß1-42-induced cell toxicity. Extracellularly applied Aß1-42 significantly increased cell surface localization of α7 ACh receptor in PC-12 cells as compared with that for non-treated control cells. Cell surface localization of α7 ACh receptor in the brain of 5xFAD mouse, an animal model of Alzheimer's disease (AD), apparently increased in an age (1-12 months)-dependent manner in association with increased accumulation of Aß1-42 in the plasma membrane component. Taken together, these results indicate that Aß1-42 promotes translocation of α7 ACh receptor towards the cell surface and that α7 ACh receptor rescues neuronal cells from Aß1-42-induced damage.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Nicotina/farmacologia , Células PC12 , Transporte Proteico/efeitos dos fármacos , Ratos , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/análiseRESUMO
Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer.
Assuntos
Adenosina/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Adenosina/efeitos adversos , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Ativação Enzimática , Humanos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
The present study investigated the effect of adipose-derived stem cell-conditioned medium (ASC-CM) on behavioral disorders in 5xFAD transgenic mice, a model of Alzheimer's disease (AD). The immobility time in the tail suspension and forced swim tests for 5xFAD mice was shorter than that for wild-type mice. Intravenous injection with ASC-CM restored the shortened immobility time for 5xFAD mice to the normal levels or to an extent, being still persistent 4 weeks after injection. ASC-CM significantly suppressed phosphorylation of Akt at Ser473 and glycogen synthase kinase 3ß (GSK-3ß) at Ser9 in the hypothalamus of 5xFAD mice, without affecting Tau phosphorylation, as compared with that for control 5xFAD mice without ASC-CM injection. ASC-CM did not affect cell surface localization of the N-methyl-d-aspartate (NMDA) receptor subunits NR1, -2A, and -2B both in the hippocampus and hypothalamus of 5xFAD mice. The results of the present study show that ASC-CM ameliorates antidepression-related behaviors in 5xFAD mice, perhaps by inhibiting Akt and activating GSK-3ß.
Assuntos
Tecido Adiposo/citologia , Doença de Alzheimer/terapia , Meios de Cultivo Condicionados , Transtornos Mentais/terapia , Células-Tronco/citologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Animais , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/metabolismo , Hipotálamo/metabolismo , Transtornos Mentais/psicologia , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) promoted GLUT4 translocation to the cell surface in differentiated 3T3-L1-GLUT4myc adipocytes through a pathway along a phosphatidylinositol 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, that mimics insulin signaling. Moreover, diDCP-LA-PE-induced GLUT4 translocation was suppressed by inhibitors of the Rho GTPase Rac1 and Rho-associated coiled-coil-containing protein kinase (ROCK) or knocking-down Rac1 and ROCK1. The results of the present study show that Rac1 and ROCK are critical for regulation of GLUT4 trafficking by diDCP-LA-PE as well as insulin.
Assuntos
Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Fosfatidiletanolaminas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/fisiologia , Adipócitos/metabolismo , Linhagem Celular , Humanos , Insulina , Fosfatidilinositol 3-Quinase/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologiaRESUMO
BACKGROUND/AIMS: The linoleic acid derivative DCP-LA selectively and directly activates PKCε. The present study aimed at understanding the mechanism of DCP-LA-induced PKCε activation. METHODS: Point mutation in the C2-like domain on PKCε was carried out, and each kinase activity was monitored in PC-12 cells using a föerster resonance energy transfer (FRET) probe with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the N- and C-terminal ends of PKCε, respectively, or in the cell-free systems using a reversed phase high-performance liquid chromatography (HPLC). Intracellular PKCε mobilization was monitored in PC-12 cells using mRuby-conjugated PKCε. DCP-LA binding to PKCε was assayed using a fluorescein conjugated to DCP-LA at the carboxyl-terminal end (Fluo-DCP). Uptake of DCP-LA into cells was measured in PC-12 ells. RESULTS: In the FRET analysis, DCP-LA decreased the ratio of YFP signal intensity/CFP signal intensity in PC-12 cells and in the cell-free kinase assay, DCP-LA increased area of phosphorylated PKC substrate peptide, indicating DCP-LA-induced PKCε activation. These effects were significantly suppressed by replacing Arg50 and Ile89 by Ala or Asn in the C2-like domain of PKCε. In the fluorescent cytochemistry, DCP-LA did not affect intracellular PKCε distribution. In the cell-free binding assay, Fluo-DCP, that had no effect on the potential for PKCε activation, bound to PKCε, and the binding was inhibited only by mutating Ile89. Extracellularly applied DCP-LA was taken up into cells in a concentration-dependent manner. Although no activation was obtained in the cell-free kinase assay, the broad PKC activator PMA activated PKCε in PC-12 cells in association with translocation towards the cell surface, which was inhibited by mutating I89A. CONCLUSION: Unlike PMA DCP-LA activates cytosolic PKCε by binding to the phosphatidylserine binding/associating sites Arg50 and Ile89, possibly at the carboxyl-terminal end and the cyclopropane rings, respectively.
Assuntos
Sítios de Ligação/efeitos dos fármacos , Caprilatos/farmacologia , Citosol/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Proteína Quinase C/metabolismo , Animais , Linhagem Celular Tumoral , Sistema Livre de Células/efeitos dos fármacos , Sistema Livre de Células/metabolismo , Citosol/metabolismo , Mutação/efeitos dos fármacos , Células PC12 , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Phosphatidylinositol (PI) regulates a variety of cell processes. The present study investigated the antitumor action of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DOPI) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DPPI) on human malignant pleural mesothelioma (MPM) cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells. METHODS: MTT assay, TUNEL staining, flow cytometry using propidium iodide (PI) and annexin V (AV), enzymatic caspase assay, and nuclear staining using DAPI were carried out, and mitochondrial membrane potentials and intracellular distribution of apoptosis-inducing factor (AIF) were monitored in cells with and without the siRNA silencing the Bid-targeted gene. RESULTS: Both DOPI and DPPI reduced cell viability for all the investigated MPM cell lines in a concentration (0.01-100 µM)-dependent manner. DOPI and DPPI significantly increased TUNEL-positive cells and the population of PI-negative/AV-positive and PI-positive/AV-positive cells, corresponding to early apoptosis and late apoptosis/secondary necrosis, respectively. DOPI and DPPI perturbed mitochondrial membrane potentials in MSTO-211H cells, but no significant activation of caspase-3, -4, -8, and -9 was obtained. DOPI and DPPI upregulated expression of Bid in MSTO-211H cells. DOPI and DPPI significantly increased nuclear localization of AIF without affecting expression of the mRNAs and proteins in MSTO-211H cells, which was inhibited by knocking-down Bid. In the DAPI staining, nuclear fragmentation and condensation were found. CONCLUSION: The results of the present study indicate that DOPI and DPPI facilitate Bid-mediated AIF release from the mitochondria, to accumulate AIF in the nucleus and induce caspase-independent apoptosis of MPM cells.
Assuntos
Fator de Indução de Apoptose/genética , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Fator de Indução de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/agonistas , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Marcação In Situ das Extremidades Cortadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatidilinositóis , Pleura/efeitos dos fármacos , Pleura/metabolismo , Pleura/patologia , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
The present study investigated 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE)-induced cell death in malignant pleural mesothelioma (MPM) cells. DAPE reduced cell viability in NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H MPM cell lines in a concentration (1-100µM)-dependent manner. In the flow cytometry using propidium iodide (PI) and annexin V (AV), DAPE significantly increased the population of PI-positive and AV-negative cells, corresponding to primary necrosis, and that of PI-positive and AV-positive cells, corresponding to late apoptosis/secondary necrosis, in NCI-H28 cells. DAPE-induced reduction of NCI-H28 cell viability was partially inhibited by necrostatin-1, an inhibitor of RIP1 kinase to induce necroptosis, or knocking-down RIP1. DAPE generated reactive oxygen species (ROS) followed by disruption of mitochondrial membrane potentials in NCI-H28 cells. DAPE-induced mitochondrial damage was attenuated by cyclosporin A, an inhibitor of cyclophilin D (CypD). DAPE did not affect expression and mitochondrial localization of p53 protein in NCI-H28 cells. DAPE significantly decreased intracellular ATP concentrations in NCI-H28 cells. Overall, the results of the present study indicate that DAPE induces necroptosis and necrosis of MPM cells; the former is mediated by RIP1 kinase and the latter is caused by generating ROS and opening CypD-dependent mitochondrial permeability transition pore, to reduce intracellular ATP concentrations.
Assuntos
Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidiletanolaminas/farmacologia , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mesotelioma/patologia , Necrose/tratamento farmacológico , Neoplasias Pleurais/patologiaRESUMO
Irinotecan, a topoisomerase I inhibitor, is clinically used as an anticancer drug. The present study investigated the anticancer effect of irinotecan on p53-negative Caco-2 and p53-positive CW2 human colorectal cancer cell lines. Cell viability for both Caco-2 and CW2 cells was little affected by treatment with irinotecan at concentrations ranging from 0.3 to 30 µmol/l for 24-48 h. Irinotecan did not increase the number of TUNEL-positive cells and did not affect the population of propidium iodide (PI)-positive and annexin V-negative cells, corresponding to primary necrosis, or that of PI-positive and annexin-positive cells, corresponding to late apoptosis/secondary necrosis, in either of the two cell lines. In the cell cycle analysis, irinotecan significantly increased the proportions at the S and G2/M phases of cell cycling in parallel with a decreased population at the G1 phase in both cell lines. Irinotecan significantly inhibited tumor growth in mice inoculated with CW2 cells. Taken together, these results indicate that irinotecan induces cell cycle arrest, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, leading to suppression of cell proliferation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Células CACO-2 , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Humanos , Irinotecano , Masculino , Camundongos Nus , Necrose , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Carga Tumoral/efeitos dos fármacosRESUMO
Naftopidil is clinically for treatment of benign prostate hyperplasia, and emerging evidence has pointed to its anticancer effect. To obtain the anticancer drug with the potential greater than that of naftopidil, we have newly synthesized the naftopidil analogue HUHS1015. The present study investigated the mechanism underlying HUHS1015-induced apoptosis of human gastric cancer cells and assessed the possibility for clinical use as an innovative anticancer drug. HUHS1015 reduced cell viability for MKN28 human well-differentiated gastric adenocarcinoma cell line and MKN45 human poorly differentiated gastric adenocarcinoma cell line in a concentration (0.3-100 µM)-dependent manner more effectively than cisplatin, a chemo-drug widely used. In the flow cytometry using propidium iodide (PI) and annexin V, HUHS1015 significantly increased the population of PI-positive and annexin V-negative cells, corresponding to primary necrosis and that of PI-positive and annexin V-positive cells, corresponding to late apoptosis/secondary necrosis, both in the two cell types. HUHS1015 significantly activated caspase-3, caspase-4, and caspase-8 in MKN45 cells, while no obvious caspase activation was found in MKN28 cells. HUHS1015 upregulated expression of the tumor necrosis factor α (TNFα) mRNA and protein in MKN45 cells, allowing activation of caspase-8 through TNF receptor and the effector caspase-3. HUHS1015 clearly inhibited tumor growth in mice inoculated with MKN45 cells, with the survival rate higher than that for the anticancer drugs cisplatin, paclitaxel, and irinotecan. The results of the present study show that HUHS1015 induces caspase-independent and caspase-dependent apoptosis of MKN28 and MKN45 human gastric cancer cells, respectively, and effectively suppresses MKN45 cell proliferation.
Assuntos
Adenocarcinoma/tratamento farmacológico , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Propanolaminas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/patologia , Compostos de Anilina/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Propanolaminas/administração & dosagem , RNA Mensageiro/metabolismo , Neoplasias Gástricas/patologia , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The newly synthesized naftopidil analogue HUHS1015 reduced viability of MKN28 and MKN45 human gastric cancer cells in a concentration (0.3-100 µM)-dependent manner, with the potential greater than that for naftopidil. In the cell cycle analysis, HUHS1015 significantly increased the proportion at the subG1 phase of cell cycling in MKN28 cells. In the flow cytometry using propidium iodide (PI) and annexin V, HUHS1015 significantly increased the populations of PI-positive/annexin V-negative and PI-positive/annexin V-positive MKN28 cells, corresponding to primary necrosis and late apoptosis/secondary necrosis, respectively. HUHS1015-induced MKN28 cell death was attenuated by the necroptosis inhibitor Nec-1. In the enzymatic caspase assay, caspase-3, -4, -8, and -9 were not sufficiently activated by HUHS1015. HUHS1015 increased nuclear localization of apoptosis-inducing factor-homologous mitochondrion-associated inducer of death (AMID), without affecting expression of the AMID mRNA and protein in MKN28 cells. HUHS1015 caused nuclear fragmentation and condensation in MKN28 cells treated with HUHS1015. Taken together, these results of the present study indicate that HUHS1015 induces both necroptosis and caspase-independent apoptosis of MKN28 cells, possibly the latter effect being due to AMID accumulation in the nucleus.
Assuntos
Compostos de Anilina/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Núcleo Celular/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Necrose/induzido quimicamente , Propanolaminas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Anexina A5/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Fase G1/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Gástricas/metabolismoRESUMO
We have originally synthesized the naftopidil analogue 1-[2-(2-methoxyphenylamino)ethylamino]-3-(naphthalene-1-yloxy)propan-2-ol (HUHS 1015) as a new anticancer drug. HUHS1015 induces cell death in a wide variety of human cancer cell lines originated from malignant pleural mesothelioma, lung cancer, hepatoma, gastric cancer, colorectal cancer, bladder cancer, prostate cancer, and renal cancer. HUHS1015-induced cell death includes necrosis (necroptosis) and apoptosis, and the underlying mechanism differs depending upon cancer cell types. HUHS1015 effectively suppresses tumor growth in mice inoculated with NCI-H2052, MKN45, or CW2 cells, with a potential similar to or higher than that of currently used anticancer drugs. Here we show how HUHS1015 might offer brilliant hope for cancer therapy.