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1.
Photodiagnosis Photodyn Ther ; 25: 29-34, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30389626

RESUMO

OBJECTIVE: To investigate the effect of photodynamic therapy (PDT) with the talaporfin sodium (mono-L-asparthyl chlorine e6: NPe-6) on human malignant meningioma cell line HKBMM cells in vitro. MATERIAL AND METHODS: After incubation with NPe6 for 4 h, cells underwent PDT (diode laser irradiation: 3.4 mW/cm2 and 1 J/cm2. Cell viability was determined in 2 malignant meningioma cell lines (human origin; HKBMM cells and rat origin; KMY-J cells) and human malignant glioma U251 cells with Cell Counting Kit-8 assay. The HKBMM cells were examined for caspase-3 activity, annexin V or propidium iodide (PI) staining, and lactate dehydrogenase leakage. Morphological change was also investigated with phase-contrast microscopy. RESULTS: In human malignant meningioma HKBMM cells, viability showed a dose- and time-dependent decrease. After 24 h of laser irradiation, NPe6 at 20 µg/ml or more induced a significant decrease in cell viability in both HKBMM cells and KMY-J cells, although they more resistance than the malignant glioma cell line U251 cells. Two kinds of morphological change were also observed in the HKBMM cells, shrinkage of the cell body, indicating apoptosis, and swelling of the cell body, indicating necrosis. In addition, both caspase-3 activity and DNA fragmentation, biochemical markers indicative of apoptosis, showed a dose-dependent increase. The percentage of necrotic cells showing positive staining for annexin V or PI was greater than that of apoptotic cells at a high concentration of NPe6. Lactate dehydrogenase leakage, a biochemical marker of necrosis, also showed a marked increase at a high concentration of NPe6. CONCLUSION: Photodynamic therapy with NPe6 induced dose- and time-dependent apoptosis in human malignant meningioma HKBMM cells. At a high concentration of NPe6, however, it induced necrosis.


Assuntos
Apoptose/efeitos dos fármacos , Meningioma/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Anexina A5/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Tamanho Celular , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Fatores de Tempo
2.
J Nat Med ; 71(1): 36-43, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27388719

RESUMO

Here, we show that AU-1, spirostanol saponin isolated from Agavaceae plants, causes a transient increase in cyclin-dependent kinase inhibitor (CDKI) p21/Cip1 through the upregulation of miRNAs, miR-34 and miR-21. AU-1 stimulated p21/Cip1 expression without exerting cytotoxicity against different types of carcinoma cell lines. In renal adenocarcinoma ACHN cells, AU-1 transiently elevated the expression level of p21/Cip1 protein without marked increases in p21/Cip1 mRNA levels. Rapid and transient increases in miR-34 and miR-21, both of which are known to upregulate p21/Cip1, were observed in AU-1-treated cells. Inhibitor for miR-34 and for miR-21 significantly blocked the AU-1-caused increase in p21/Cip1, indicating that elevation of p21/Cip1 protein by AU-1 is dependent on these microRNAs. We further clarified that NAD-dependent deacetylase SIRT1, a direct target of miR-34, is decreased by the treatment with AU-1. Furthermore, we found that SIRT1-knockdown increases p21/Cip1 protein levels in an miR-21-dependent manner. On the other hand, ectopic expression of p21/Cip1 resulted in the lowered expression of miR-34 and miR-21, suggesting that reciprocal regulation exists between p21/Cip1 and these miRNAs. We propose that the following feedback network composed of miR-34/SIRT1/miR-21/p21 is triggered by the treatment with AU-1: in cells treated with AU-1, transient elevation of miR-34 leads to the downregulation of SIRT1, thereby miR-21 is freed from SIRT1-dependent suppression. Then, elevated miR-21 upregulates p21/Cip1 protein, followed by the suppression of miR-34 expression.


Assuntos
Ampicilina/análogos & derivados , Asparagaceae/química , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ampicilina/uso terapêutico , Carcinoma de Células Renais , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes , Regulação para Baixo , Células Hep G2 , Humanos , RNA Mensageiro/biossíntese
3.
Biol Pharm Bull ; 38(9): 1420-4, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26328500

RESUMO

Although macrophage phagocytoses modified low-density lipoprotein (LDL), excessive accumulation of modified LDL induces macrophage foam cell formation, which is a feature of atherosclerotic plaque. Thus, the identification of scavenger receptor for modified LDL will provide better understanding of an atherosclerotic event. We recently showed that nucleolin expressed on macrophages acts as a scavenger receptor for various endogenous discarded products. Here, we investigated whether or not nucleolin is involved in the uptake of acetylated LDL (AcLDL). In contrast to normal LDL, AcLDL directly bound to immobilized nucleolin. AcLDL exhibited a higher affinity for macrophages than normal LDL. This binding of AcLDL was inhibited by anti-nucleolin antibody and antineoplastic guanine-rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer. In addition, AcLDL exhibited a higher affinity for HEK cells transfected with nucleolin than those without. Further, intracellular accumulation of AcLDL was also inhibited by anti-nucleolin antibody. The results of this study suggest that nucleolin expressed on macrophages is a receptor for AcLDL.


Assuntos
Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Depuradores/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Nucleolina
4.
Lasers Med Sci ; 30(6): 1739-45, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26109138

RESUMO

Photodynamic therapy (PDT) using photosensitizer induces several types of cell death, such as apoptosis, necrosis, and autophagy, depending on the PDT procedure, photosensitizer type, and cell type. We previously demonstrated that PDT using the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6; NPe6-PDT) induces both mitochondrial apoptotic and necrotic cell death in human glioblastoma T98G cells. However, details regarding the mechanism of necrosis caused by NPe6-PDT are unclear. Here, we investigated whether or not necroptosis, a recently suggested form of programmed necrosis, is involved in the necrotic cell death of NPe6-PDT-treated T98G cells. Leakage of lactate dehydrogenase (LDH) from the cell layer into conditioned medium was significantly increased by NPe6 (25 and 50 µg/ml)-PDT, indicating that NPe6-PDT induces necrosis in these cells. NPe6 (25 µg/ml)-PDT treatment also induced conversion of microtubule-associated protein 1 light-chain 3 (LC3)-I into phosphatidylethanolamine-conjugated LC3-II accompanying autophagosome formation, indicators of autophagy; however, of note, NPe6 (50 µg/ml)-PDT did not induce such autophagic changes. In addition, both necrostatin-1 (a necroptosis inhibitor) and knockdown of necroptotic pathway-related proteins [e.g., receptor interacting serine-threonine kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] inhibited leakage of LDH caused by NPe6 (25 µg/ml)-PDT. Taken together, the present findings revealed that NPe6-PDT-induced necrotic cell death is mediated in part by the necroptosis pathway in glioblastoma T98G cells.


Assuntos
Apoptose/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Fotoquimioterapia/métodos , Porfirinas/uso terapêutico , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Clorofilídeos , Humanos , L-Lactato Desidrogenase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Necrose , Fagossomos/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/farmacologia
5.
Biol Pharm Bull ; 38(1): 116-21, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25744467

RESUMO

Scavenger receptors have a broad range of functions that include pathogen clearance, and identification of the scavenger receptor family has been of great benefit to the field of physiology. The shuttling-protein nucleolin has recently been shown to possess scavenger receptor-like activity. We therefore investigated whether or not nucleolin is a receptor for maleylated-bovine serum albumin (maleylated-BSA), which is a common ligand for scavenger receptors. Binding and phagocytosis of native control-BSA by thioglycollate-elicited mouse peritoneal macrophages was weak, but that of maleylated-BSA was strong. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with maleylated-BSA but not control-BSA or maleic anhydride. Further, co-treatment of macrophages with anti-nucleolin antibody, but not control-immunoglobulin G, inhibited binding of maleylated-BSA. In addition, antineoplastic guanine rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer, inhibited binding of maleylated-BSA. Further, binding of maleylated-BSA to nucleolin-transfected HEK293 cells was higher than that by control HEK cells. These results indicate that nucleolin is a receptor that enables macrophages to recognize maleylated-BSA.


Assuntos
Macrófagos Peritoneais/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Células HEK293 , Humanos , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Fagocitose , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Nucleolina
6.
J Toxicol Sci ; 39(6): 821-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25374373

RESUMO

While photodynamic therapy (PDT) is an effective treatment for glioma, induction of apoptotic cell death of glioma cells is important for ensuring efficacy and safety of PDT treatment in glioma patients, as necrotic cell death can induce late appearance of obstacles in treatment. Here, we investigated the relationship between type of cell death and PDT treatment conditions involved in laser and photosensitizer dosage in human glioblastoma T98G cells. Photosensitizer talaporfin sodium-mediated PDT (NPe6-PDT) treatment induced laser and NPe6 dose-dependent cell death in T98G cells, whereas almost all cells pretreated with NPe6 at ≥ 30 µg/mL were killed by laser irradiation, regardless of laser dose. Morphological analysis showed that combination of high doses of NPe6 and laser irradiation changes the dominant cell death process from apoptosis to necrosis. Biochemical analysis (detection of caspase-3 activity and staining of cell surface-exposed phosphatidylserine) also showed that increasing laser dose changes the type of cell death from apoptotic to necrotic cell death after high-dose treatment with NPe6. Lactate dehydrogenase leakage assay demonstrated that a laser dose of 5 J/cm(2) induced less leakage than 30 J/cm(2). Our results suggested that type of glioma cell death in NPe6-PDT changed with fluctuations in laser and NPe6 dose, and that combination of 30 µg/mL NPe6 with 5 J/cm(2) laser is the best treatment condition for inducing an increase in apoptotic cells while keeping rate of necrotic cell death low in this in vitro study.


Assuntos
Antineoplásicos/toxicidade , Glioblastoma/patologia , Lasers , Fotoquimioterapia , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/toxicidade , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Glioblastoma/tratamento farmacológico , Humanos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/uso terapêutico , Doses de Radiação , Células Tumorais Cultivadas
7.
Photodiagnosis Photodyn Ther ; 11(4): 556-64, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25262961

RESUMO

BACKGROUND: Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. We have previously reported that PDT induces apoptotic cell death in glioma cells when the photosensitizer talaporfin sodium (NPe6) is used. Here, we investigated the combined effect of NPe6-PDT with temozolomide, a DNA-alkylating drug used in glioma therapy. METHODS: Human glioblastoma T98G cells and human glioma U251 cells were used as glioma cells. Cell viability was evaluated by WST-8 assay. Apoptosis was evaluated by measurement of caspase-3 activity and DNA-fragmentation. Intracellular reactive oxygen species were evaluated by dihydrorhodamine assay. RESULTS: While the degree of NPe6-PDT induced cell death unchanged in T98G and U251 cells when temozolomide treatment was adjuvant, it was dose-dependently increased by concomitant treatment with temozolomide. Further, concomitantly administered temozolomide dose-dependently increased caspase-3 activity and DNA-fragmentation, while adjuvant-temozolomide did not. These results are suggesting that concomitantly administered temozolomide potentiates the effect of NPe6-PDT to facilitate apoptotic cell death. Additionally, concomitantly administered temozolomide increased intracellular NPe6-fluorescence and reactive oxygen species, suggesting that the augmentation effect of combined treatment may be due to increased intracellular accumulation of NPe6. CONCLUSION: These results suggest that concomitant treatment with NPe6-PDT and temozolomide is a potentially useful therapy for glioma.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Fotoquimioterapia/métodos , Porfirinas/administração & dosagem , Antineoplásicos Alquilantes/administração & dosagem , Apoptose/efeitos da radiação , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Dacarbazina/administração & dosagem , Quimioterapia Combinada , Glioma/patologia , Humanos , Fármacos Fotossensibilizantes/administração & dosagem , Temozolomida , Resultado do Tratamento
8.
Biol Pharm Bull ; 37(4): 588-96, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24818254

RESUMO

Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/metabolismo , Animais , Aptâmeros de Nucleotídeos , Bovinos , Gliceraldeído/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Oligodesoxirribonucleotídeos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Soroalbumina Bovina/metabolismo , Transfecção , Nucleolina
9.
Biol Pharm Bull ; 36(10): 1587-93, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23912744

RESUMO

Amyloid-beta peptide 1-42 (Aß42) plays a key role in the neurotoxicity found in Alzheimer's disease. Mononuclear phagocytes in the brain (microglia), can potentially clear Aß via phagocytosis. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether this receptor interacts specifically with Aß type 1-42 and mediates its phagocytosis by microglia. While monomeric and fibril Aß42 were phagocytosed by mouse microglial EOC2 cells, amyloid ß peptide 1-40 (Aß40) was only weakly phagocytosed. Surface plasmon-resonance analysis revealed that nucleolin strongly associates with Aß42, but only weakly associates with Aß40. Immunofluorescence staining of anti-nucleolin antibody revealed that EOC2 cells and rat primary microglia express nucleolin on their cell surfaces. Further, pretreating EOC2 cells with anti-nucleolin antibody, but not immunoglobulin G (IgG), inhibited phagocytosis of monomeric Aß42 by microglia. Additionally, nucleolin-transfected HEK293 cells phagocytosed monomeric and fibril Aß42 but not monomeric and fibril Aß40. Moreover, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited phagocytosis of monomeric and fibril Aß42, but not monomeric and fibril Aß40. These results indicate that nucleolin is a receptor that allows microglia to recognize monomeric and fibril Aß42.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Microglia/metabolismo , Sistema Fagocitário Mononuclear , Fagocitose , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Doença de Alzheimer/imunologia , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Ratos , Nucleolina
10.
Photodiagnosis Photodyn Ther ; 10(2): 103-10, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23769275

RESUMO

OBJECTIVE: To investigate the kinetics of cell death in human glioma cell lines induced by photodynamic therapy (PDT) with the second-generation photosensitizer talaporfin sodium (TS) and a 664-nm diode laser. MATERIALS AND METHODS: Three human glioma cell lines (T98G, A172, U251) were studied. After incubation of the cell lines with various concentrations of TS for 4 h, PDT using diode laser irradiation at 33 mW/cm² and 10 J/cm² was performed. Cell viability and changes in cell morphology were examined by the Cell Counting Kit-8 assay and phase-contrast microscopy, respectively. In addition, to evaluate the pathology of cell death, changes in cell viability after treatment with a caspase activation inhibitor and an autophagy inhibitor were also examined. RESULTS: In all 3 human glioma cell lines, TS induced dose-dependent cell death. However, the 50% lethal dose of TS varied among these cell lines. The main morphological feature of cell death was shrinkage of the cell body, and the number of cells with this morphological change increased in a time-dependent manner, resulting in cell death. In addition, a dose-dependent improvement in cell viability by the caspase inhibitor Z-VAD-fmk was observed. CONCLUSION: PDT with TS induces dose-dependent apoptosis in human glioma cell lines. However, the sensitivity to PDT varied among the cell lines, indicating a possible difference in the intracellular content of TS, or a difference in the susceptibility to the intracellular oxidative stress caused by PDT.


Assuntos
Apoptose/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/patologia , Fotoquimioterapia/métodos , Porfirinas/administração & dosagem , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Radiossensibilizantes/administração & dosagem , Resultado do Tratamento
11.
Cell Struct Funct ; 38(1): 43-54, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23400223

RESUMO

Macrophages remove deteriorating cells (those undergoing apoptosis and oxidation) via poly-N-acetyllactosaminyl chains on CD43 caps, a major cell-surface glycoprotein. Unusually high intracellular calcium levels are also deteriorating for cells and tissue. Here we artificially elevated calcium levels in cells and examined the mechanism by which this elevation was resolved by macrophages. Results showed that treatment with the calcium ionophore A23187 and ionomycin induces capping of CD43 on Jurkat cells, which are subsequently recognized and phagocytosed by macrophages, indicating that macrophages regard cells with elevated calcium as targets for removal. Further tests showed that A23187- and ionomycin-treated Jurkat cells did not induce apoptotic changes such as DNA fragmentation or phosphatidylserine expression, indicating that these cells were removed despite still being viable. Jurkat cells pretreated with anti-CD43 antibody or those with poly-N-acetyllactosaminyl chains containing oligosaccharides inhibited macrophage binding, indicating that macrophages recognize the poly-N-acetyllactosaminyl chains on CD43. Binding was also inhibited by treating macrophages with anti-nucleolin antibody, indicating that recognition occurs through nucleolin, a cell-surface receptor. Further, nucleolin-transfected HEK293 cells bound A23187-treated cells, and this binding was inhibited by in the presence of oligosaccharides. Taken together, these results show that elevated calcium levels induce CD43 capping, and macrophages remove the cells if their nucleolin receptors can bind to the poly-N-acetyllactosaminyl chains of capped CD43.


Assuntos
Apoptose , Cálcio/farmacologia , Leucossialina/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Calcimicina/farmacologia , Cálcio/metabolismo , Carboidratos/química , Fragmentação do DNA/efeitos dos fármacos , Células HEK293 , Humanos , Ionomicina/farmacologia , Células Jurkat , Leucossialina/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Oligossacarídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Nucleolina
12.
Biol Pharm Bull ; 36(2): 215-21, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23196427

RESUMO

Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos c/metabolismo , Fragmentação do DNA , Glioma/metabolismo , Glioma/patologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Necrose/induzido quimicamente , Necrose/metabolismo , Necrose/patologia
13.
Biosci Biotechnol Biochem ; 76(11): 2068-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23132587

RESUMO

The mechanism was investigated for macrophage recognition of cells oxidized by diamide, a thiol group-specific oxidizing reagent. Jurkat cells exposed to various concentrations of diamide were recognized by macrophages, the cells exposed to 25 µM diamide being best recognized. CD43, a major glycoprotein on the Jurkat cell surface, tended to form clusters upon diamide oxidization, and pretreating Jurkat cells with the anti-CD43 antibody inhibited macrophage binding. This indicates that macrophages appeared to recognize CD43. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and a Western blot analysis of CD43 of the diamide-oxidized cells showed no increase in the amount of cross-linked CD43 compared with control cells, indicating that cross-linking of CD43 by a disulphide bond was not involved in the clustering. Both CD43 clustering and binding of the oxidized cells to macrophages was prevented by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), suggesting that the oxidized and macrophage-bound cells were undergoing apoptosis. A closer examination revealed that the caspase-3 activity, chromatin condensation, and DNA fragmentation in Jurkat cells were all increased by oxidation. The macrophage receptor involved in the binding appeared to be the cell-surface protein, nucleolin; an anti-nucleolin antibody treatment inhibited the binding. These results suggest that thiol group-oxidized cells underwent early apoptosis and were recognized by nucleolin on macrophages as early apoptotic cells.


Assuntos
Apoptose , Metabolismo dos Carboidratos , Macrófagos/citologia , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Compostos de Sulfidrila/metabolismo , Apoptose/efeitos dos fármacos , Metabolismo dos Carboidratos/efeitos dos fármacos , Diamida/farmacologia , Humanos , Células Jurkat , Ligantes , Macrófagos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Nucleolina
14.
Biol Pharm Bull ; 35(4): 551-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22466560

RESUMO

Apoptotic cells must be recognized early for phagocytosis to ensure that their toxic contents do not damage neighboring cells. In some cases this is achieved via CD43-capped membrane glycoproteins, the sialylpolylactosaminyl chains of which serve as ligands for phagocytosis by macrophages. However, because many additional changes occur during apoptosis, determining exactly which events are responsible for signaling macrophages to initiate phagocytosis remains a challenge. Here, we examined one clearance mechanism in detail and determined that capping of CD43 alone is sufficient to initiate phagocytosis. We induced macrophage-mediated phagocytosis by using cytochalasin B to artificially cap CD43 on healthy (non-apoptotic) Jurkat cells. Additional experiments confirmed that sialylpolylactosaminyl chains formed through this capping method are a prerequisite for removal, and that nucleolin is the macrophage receptor responsible for their detection. These findings strongly suggest that capping of CD43 presents a sufficient signal for phagocytosis without any additional membrane changes.


Assuntos
Citocalasina B/farmacologia , Leucossialina/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Apoptose , Células HEK293 , Humanos , Células Jurkat , Fosfoproteínas/imunologia , Proteínas de Ligação a RNA/imunologia , Nucleolina
15.
Biol Pharm Bull ; 32(4): 564-72, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19336885

RESUMO

The mechanism of macrophage recognition of oxidatively damaged cells was investigated. Jurkat T cells exposed to various concentrations of H(2)O(2) were bound and phagocytosed by macrophages. The cells exposed to 0.1 mM H(2)O(2) were best bound. The cell-surface ligands recognized by macrophages were suggested to be sialylpolylactosaminyl sugar chains of a major sialoglycoprotein CD43 because 1) the cell binding was inhibited by oligosaccharides containing sialylpolylactosaminyl chains, and their inhibitory activity was destroyed by a polylactosamine-cleaving enzyme endo-beta-galactosidase, and by neuraminidase; 2) the oxidized Jurkat cells pretreated with either glycosidase or with anti-CD43 antibody were not bound. The macrophage receptor involved in the binding was suggested to be cell-surface nucleolin because 1) anti-nucleolin antibody inhibited the binding; 2) nucleolin-transfected HEK293 cells bound the oxidized cells; and 3) this binding was inhibited by anti-nucleolin antibody and by anti-CD43 antibody. CD43 on oxidized Jurkat cells tended to form clusters in good accordance with their susceptibility to the macrophage binding. CD43 clustering and the oxidized-cell binding to macrophages were prevented by a caspase inhibitor Z-VAD-fmk, suggesting that the oxidized and bound cells were undergoing apoptosis. Indeed, caspase-3 activity of Jurkat cells increased by the oxidation. These results suggest that moderately oxidized cells undergo apoptosis and are recognized by macrophages as early apoptotic cells.


Assuntos
Apoptose/fisiologia , Glicoproteínas/química , Macrófagos/fisiologia , Estresse Oxidativo/fisiologia , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , Caspases/metabolismo , Linhagem Celular , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Citosol/fisiologia , Citosol/ultraestrutura , Humanos , Células Jurkat , Leucossialina/química , Proteínas de Membrana/química , Oxirredução , Nucleolina
16.
Biochem Biophys Res Commun ; 363(1): 57-62, 2007 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17854772

RESUMO

Human erythrocytes exposed to appropriate concentrations of H(2)O(2) for 1h became susceptible to the binding and phagocytosis by macrophages. The binding was inhibited by anti-band 3 serum and prevented by pretreatment of erythrocytes with a polylactosamine-cleaving enzyme endo-beta-galactosidase, indicating that polylactosaminyl sugar chains of band 3 are recognized by macrophages. The macrophage receptor involved was suggested to be nucleolin, a recently identified macrophage surface protein recognizing sialylpolylactosaminyl-chain clusters on early apoptotic cells, because anti-nucleolin antibody and a soluble form of recombinant nucleolin blocked the recognition. Treatment of erythrocytes with caspase inhibitors Z-VAD-fmk or Z-DQMD-fmk (caspase 3 selective) before the oxidation resulted in lowered binding of the oxidized erythrocytes to macrophages, suggesting that actions of caspases, particularly those of caspase 3, are prerequisite for the membrane changes leading to band 3 aggregation. Moreover, the cytosolic caspase 3 was found to be activated by H(2)O(2), and the extent of the activation correlated well with the susceptibility of the oxidized erythrocytes to the macrophage recognition. These results suggest that oxidative stress renders the erythrocytes susceptible to clearance by macrophages through activation of caspases leading to band 3 aggregation.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Caspases/metabolismo , Eritrócitos/fisiologia , Peróxido de Hidrogênio/administração & dosagem , Macrófagos/fisiologia , Fagocitose/fisiologia , Transdução de Sinais/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Taxa de Depuração Metabólica/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fagocitose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
17.
J Biol Chem ; 280(47): 39284-93, 2005 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-16135517

RESUMO

Early apoptotic Jurkat T cells undergo capping of CD43, and its polylactosaminyl saccharide chains serve as ligands for phagocytosis by macrophages. This suggests the presence of a polylactosaminoglycan-binding receptor on macrophages. Here we show that this receptor is nucleolin, a multifunctional shuttling protein present in nucleus, cytoplasm, and on the surface of some types of cells. Nucleolin was detected at the surface of macrophages, and anti-nucleolin antibody inhibited the binding of the early apoptotic cells to macrophages. Nucleolin-transfected HEK293 cells expressed nucleolin on the cell surface and bound the early apoptotic cells but not phosphatidylserine-exposing late apoptotic cells. This binding was inhibited by anti-nucleolin antibody, by polylactosamine-containing oligosaccharides, and by anti-CD43 antibody. Deletion of the antibody binding region of nucleolin resulted in loss of the apoptotic cell-binding ability. Moreover, truncated recombinant nucleolin in solution containing this region blocked the apoptotic cell binding to macrophages, and the blocking effect was cancelled by the oligosaccharides. These results indicate that nucleolin is a macrophage receptor for apoptotic cells.


Assuntos
Apoptose/fisiologia , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Humanos , Técnicas In Vitro , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Monócitos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Nucleolina
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