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1.
Br J Radiol ; 93(1108): 20190792, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31939310

RESUMO

OBJECTIVES: To evaluate the feasibility and optimal restricted angle of the complete-directional-complete block (CDCB) technique in helical tomotherapy (HT) by including regional nodal irradiation (RNI) with the internal mammary node (IMN) in left-sided breast cancer. METHODS: Ten left-sided breast cancer patients treated with 50 Gy in 25 fractions were compared with five-field intensity-modulated radiation therapy (5F-IMRT) and six types of HT plans. In the HT plans, complete block (CB), organ-based directional block (OBDB) and CDCB with different restricted angles were used. RESULTS: The conformity index (CI) between the CDCB0,10,15,20 and 5F-IMRT groups was similar. Compared to CB, OBDB and 5F-IMRT, CDCB20 resulted in a decreased ipsilateral mean lung dose. The low-dose region (V5) of the ipsilateral lung in OBDB (84.0%) was the highest among all techniques (p < 0.001). The mean dose of the heart in CB was significantly reduced (by 11.5-22.4%) compared with other techniques. The V30 of the heart in CDCB20 (1.9%) was significantly lower than that of CB, OBDB and 5F-IMRT. Compared to the mean dose of the left anterior descending (LAD) artery of 5F-IMRT (27.0 Gy), CDCB0, CDCB10, CDCB15, CDCB20 and OBDB reduced the mean dose effectively by 31.7%, 38.3%, 39.6%, 42.0 and 56.2%, respectively. Considering the parameters of the organs-at-risk (OARs), CDCB10,15,20 had higher expectative values than the other techniques (p = 0.01). CONCLUSIONS: HT with the CDCB technique is feasible for treating left-sided breast cancer patients. The CDCB10-20 techniques not only achieved similar planning target volume coverage, homogeneity and dose conformity but also allowed better sparing of the heart and bilateral lungs. ADVANCES IN KNOWLEDGE: For left-sided breast cancer patients whose RNI field includes the IMN, heart avoidance is an important issue. The CDCB technique achieved good PTV coverage, homogeneity and dose conformity and allowed better sparing of the mean dose of the lung, the LAD artery, and the heart and reduced the V30 of the heart.


Assuntos
Coração/efeitos da radiação , Pulmão/efeitos da radiação , Irradiação Linfática/métodos , Órgãos em Risco/efeitos da radiação , Lesões por Radiação/prevenção & controle , Radioterapia de Intensidade Modulada/métodos , Neoplasias Unilaterais da Mama/radioterapia , Fracionamento da Dose de Radiação , Estudos de Viabilidade , Feminino , Humanos , Órgãos em Risco/diagnóstico por imagem , Planejamento da Radioterapia Assistida por Computador/métodos , Neoplasias Unilaterais da Mama/diagnóstico por imagem
2.
J Mol Med (Berl) ; 97(9): 1345-1357, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31302714

RESUMO

Corneal endothelial cell (CEC) dysfunction causes corneal edema that may lead to blindness. In addition to corneal transplantation, simple descemetorhexis has been proposed to treat centrally located disease with adequate peripheral cell reserve, but promoting the centripetal migration of CECs is pivotal to this strategy. Here, we show that targeting non-muscle myosin II (NMII) activity by Y27632, a ROCK inhibitor, or blebbistatin, a selective NMII inhibitor, promotes directional migration of CECs and accelerates in vitro wound healing. The lamellipodial protrusion persistence is increased, and actin retrograde flow is decreased after NMII inhibition. Counteracting lamellipodial protrusion by actin-related protein 2/3 (ARP2/3) inhibitor abolishes this migration-promoting effect. Although both Y27632 and blebbistatin accelerate wound healing, cell junctional integrity and barrier function are better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model. Our findings indicate that NMII is a promising therapeutic target in the treatment of CEC dysfunction. KEY MESSAGES: NMII inhibition promotes directional migration and wound healing of CECs in vitro. Lamellipodial protrusion persistence is increased after NMII inhibition. Selective NMII inhibitor preserves junctional integrity better than ROCK inhibitor. Selective NMII inhibitor accelerates corneal deturgescence after wounding in vivo.

3.
Radiat Oncol ; 14(1): 90, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146741

RESUMO

BACKGROUND: To evaluate the practicality of NS-21 cream with regard to its skin-related toxicity in patients with head and neck cancer (HNC) who are undergoing concurrent chemoradiation therapy (CCRT) or radiotherapy (RT). METHODS: Between July 2015 and November 2017, 30 HNC patients who underwent RT or CCRT were randomly allocated to receive either NS-21 or control treatment on their irradiated skin three times per day, starting at the initiation of RT or CCRT and ending 2 weeks after the completion of RT or until the appearance of grade 3 acute radiation dermatitis (ARD). Dermatitis was recorded weekly according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Skin humidity was monitored by a digital moisture meter. The generalized estimating equation (GEE) and logit link function method were used for statistical analysis. RESULTS: No serious adverse events were observed in either group. Itching dermatitis occurred on the right lower neck in one patient of the NS-21 group during the 3rd week of CCRT, but the severity was mild. The median skin moisture value at the time of the final treatment was significantly different between the study and control groups (30.6 vs. 27.3, p = 0.013). Additionally, there was an inverse relationship between skin moisture and ARD grade (B = -0.04, p = 0.005). The incidence of ARD at the time of the last treatment was not significantly different between the study and control groups (6.7% vs 26.7%, p = 0.165). The risk of grade 3 ARD for skin that had received an irradiation dose of 47-70 Gy was higher than that of skin that had received an irradiation dose ≤46 Gy (OR = 31.06, 95% CI =5.95-162.21, p < 0.001). Nevertheless, the risk of ARD was not significantly different between the groups (OR = 0.38, 95% CI = 0.08-1.74, p = 0.212). CONCLUSIONS: NS-21 was well tolerated and effective for the maintenance of skin moisture; however, there was no statistically significant reduction in the risk of ARD in HNC patients undergoing RT or CCRT when compared with HNC patients in the control group. TRIAL REGISTRATION: The study was approved by the Institutional Review Board of Far Eastern Memorial Hospital ( FEMH-IRB , 104048-F), Registered 1st June 2015.


Assuntos
Fármacos Dermatológicos/uso terapêutico , Neoplasias de Cabeça e Pescoço/radioterapia , Fenilacetatos/uso terapêutico , Radiodermatite/prevenção & controle , Adulto , Idoso , Idoso de 80 Anos ou mais , Fármacos Dermatológicos/administração & dosagem , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fenilacetatos/administração & dosagem , Radiodermatite/patologia , Dosagem Radioterapêutica , Radioterapia de Intensidade Modulada , Resultado do Tratamento
4.
Cornea ; 38(3): 360-363, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30601173

RESUMO

PURPOSE: To evaluate the safety and feasibility of patent blue (PB) as the vital dye in Descemet membrane endothelial keratoplasty (DMEK). METHODS: Bovine corneal endothelial cells were incubated with different concentrations (0.02%-2.5%) of PB. The cell viability, which was assessed by Cell Counting Kit-8 assay, was compared with that of untreated control and 0.06% to 0.4% trypan blue. The dyes were also used for graft preparation and implantation in the porcine eye model to evaluate stain quality, dye retention, and the feasibility of using PB in DMEK surgery. RESULTS: No obvious increase in cytotoxicity was detected for 0.06% to 0.4% trypan blue and PB at concentrations up to 1.0%, but the cell viability after incubating with 1.5% to 2.5% PB was significantly reduced. PB at 0.5% to 1.0% generated good staining quality that can be used to facilitate graft implantation. Although the staining quality of 0.5% to 1.0% PB faded to an intermediate level after a 30-minute wash in phosphate-buffered saline, dye retention persisted for up to 24 hours. CONCLUSIONS: PB at 0.5% to 1.0% is biocompatible and can stain the graft sufficiently, making it an alternative for DMEK surgery.


Assuntos
Corantes/toxicidade , Perda de Células Endoteliais da Córnea/patologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Células Endoteliais/efeitos dos fármacos , Epitélio Posterior/efeitos dos fármacos , Corantes de Rosanilina/toxicidade , Coloração e Rotulagem/métodos , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Suínos , Azul Tripano/toxicidade
5.
Oncologist ; 23(12): 1426-1435, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29728468

RESUMO

BACKGROUND: The purpose of this study was to review the risks and benefits of concurrent chemoradiation therapy (CCRT) with esophageal self-expandable metal stents (SEMS) for the treatment of locally advanced esophageal cancer. MATERIALS AND METHODS: Between January 2014 and December 2016, the data from 46 locally advanced esophageal cancer patients who received CCRT at our institution were retrospectively reviewed. Eight patients who received CCRT concomitant with SEMS placement (SEMS plus CCRT group) and thirty-eight patients who received CCRT without SEMS placement (CCRT group) were identified. The risk of developing esophageal fistula and the overall survival of the two groups were analyzed. RESULTS: The rate of esophageal fistula formation during or after CCRT was 87.5% in the SEMS plus CCRT group and 2.6% in the CCRT group. The median doses of radiotherapy in the SEMS plus CCRT group and the CCRT group were 47.5 Gy and 50 Gy, respectively. SEMS combined with CCRT was associated with a greater risk of esophageal fistula formation than CCRT alone (hazard ratio [HR], 72.30; 95% confidence interval [CI], 8.62-606.12; p < .001). The median overall survival times in the SEMS plus CCRT and CCRT groups were 6 months and 16 months, respectively. Overall survival was significantly worse in the SEMS plus CCRT group than in the CCRT group (HR, 5.72; 95% CI, 2.15-15.21; p < .001). CONCLUSION: CCRT concomitant with SEMS for locally advanced esophageal cancer results in earlier life-threatening morbidity and a higher mortality rate than treatment with CCRT alone. Further prospective and randomized studies are warranted to confirm these observations. IMPLICATIONS FOR PRACTICE: Patients treated with SEMS placement followed by CCRT had higher risk of esophageal fistula formation and inferior overall survival rate compared with patients treated with CCRT alone. SEMS placement should be performed cautiously in patients who are scheduled to receive CCRT with curative intent.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Terapia Combinada/métodos , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/radioterapia , Stents/normas , Idoso , Quimiorradioterapia/métodos , Neoplasias Esofágicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
6.
Exp Eye Res ; 124: 86-92, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24858696

RESUMO

Intraoperative mitomycin C (MMC) is widely used to prevent pterygium recurrence and glaucoma filtering bleb failure, but it has been shown to induce corneal inflammation and cell death. Postoperative dexamethasone (DEX) is advocated to reduce MMC-related inflammation and cell death in corneal fibroblasts. Nevertheless, its long-term regulation mechanism in Tenon's capsule remains to be explored. The purpose of this study was to investigate how DEX modifies MMC's effects in human Tenon's capsule fibroblasts (HTFs). HTFs isolated from the pterygium surgical patients (n = 6) were treated with MMC at 0, 0.1, 0.2, and 0.4 mg/ml for 5 min and incubated in DEX at 10 µM for 0, 1, 2, and 3 days. Recombinant interleukin-8 (IL-8) was used to verify the effect of MMC-related IL-8 secretion. Cell proliferation of all the treated cells was analyzed by WST-1 assay. The amount of IL-8 secretion in HTFs was determined by enzyme-linked immunosorbent assay. Immunoblotting assay was used to analyze the expression of peroxisome-proliferator-activated receptor gamma (PPARγ) and B-cell lymphoma-extra large (Bcl-xL). Our results revealed that MMC significantly reduced the HTF cell proliferation rate. Additionally, MMC significantly upregulated IL-8 secretion in HTFs concentration-dependently. At 3 days post treatment (dpt), 5-min exposures to 0.1, 0.2, and 0.4 mg/ml MMC resulted in 1.4-fold (p = 0.012), 1.6-fold (p = 0.012), and 2.5-fold (p = 0.001) increases of IL-8 secretion. In contrast, DEX reversed the MMC-retarded cell proliferation rate (p = 0.036) and repressed MMC-related IL-8 secretion by 33.5% at 3 dpt (p = 0.003). Addition of recombinant IL-8 noticeably suppressed HTF cell proliferation in a concentration-dependent manner. DEX stimulated upregulation of both PPARγ and Bcl-xL at 1 dpt in normal HTFs and at 2 dpt in MMC-treated HTFs. PPARγ silencing reduced expression of PPARγ and Bcl-xL, but enhanced IL-8 secretion (p < 0.001). On the other hand, Bcl-xL silencing enhanced IL-8 secretion (p < 0.001), but did not affect PPARγ expression. These revealed that IL-8 secretion in HTFs is modulated by PPARγ-dependent Bcl-xL signaling. We conclude that DEX reversed the MMC-inhibited HTF cell proliferation via diminishing the MMC-induced IL-8 secretion, which resulted from a late-phase upregulation of the PPARγ and Bcl-xL. These long-term effects suggest a beneficial postoperative DEX treatment following intraoperative MMC application.


Assuntos
Dexametasona/farmacologia , Fibroblastos/metabolismo , Interleucina-8/metabolismo , Pterígio/tratamento farmacológico , Cápsula de Tenon/metabolismo , Apoptose , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Interleucina-8/efeitos dos fármacos , Mitomicina/farmacologia , PPAR gama/biossíntese , PPAR gama/genética , Pterígio/metabolismo , Pterígio/patologia , RNA/genética , Cápsula de Tenon/patologia , Proteína bcl-X/biossíntese , Proteína bcl-X/genética
7.
Br J Ophthalmol ; 95(2): 277-83, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21036788

RESUMO

BACKGROUND/AIMS: Povidone-iodine (PI) is commonly used as a preoperative disinfectant; however, it has been shown to be cytotoxic. The present study was performed to investigate the mechanism by which PI causes cell death. METHODS: Primary human corneal fibroblasts (HCF) and a human corneal epithelial cell line (HCEC) were treated with 0.1-5% PI for 1 min. Cell morphology and growth were examined by phase-contrast microscopy and genomic DNA quantification. Cellular enzyme activities were detected by water-soluble tetrazolium salt (WST-1) and calcein-acetoxymethylester staining, whereas membrane integrity was determined by a membrane-impermeable dye. The cell fixation effect of PI was assayed by analysis of genomic DNA integrity and resistance to ionic detergent SDS lysis. The interleukin-8 (IL-8) secretion after adding interleukin-1ß (IL-1b) or lipopolysaccharide (LPS) was determined by ELISA. RESULTS: PI treatment inhibited HCF and HCEC cell growth without changing cellular morphology; however, cells became resistant to SDS lysis. The mitochondrial dehydrogenase and intracellular esterase activities as well as cell membrane integrity were abolished by PI treatment. Genomic DNA integrity from PI-treated groups was similar to that from alcohol-fixed groups. IL-1b- and LPS-induced IL-8 secretion was abolished by PI treatment. CONCLUSIONS: Where PI concentration is sufficient to cause cell death, this occurs through fixation rather than necrosis in cultured human corneal stromal and epithelial cell.


Assuntos
Anti-Infecciosos Locais/farmacologia , Morte Celular , Córnea/citologia , Fibroblastos/efeitos dos fármacos , Povidona-Iodo/farmacologia , Anti-Infecciosos Locais/efeitos adversos , Células Cultivadas , Fibroblastos/fisiologia , Humanos , Povidona-Iodo/efeitos adversos
8.
Wound Repair Regen ; 18(1): 59-69, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20002897

RESUMO

The purpose of this study was to investigate the effect of dexamethasone (DEX) on mitomycin C (MMC)-induced inflammatory cytokine expression in corneal fibroblasts. Primary human corneal fibroblasts were treated with MMC, dexamethasone, or in combination. Morphological changes and cell growth were documented using phase-contrast microscopy and PicoGreen assay, respectively. Cell apoptosis was evaluated by annexin V/propidium iodide staining, whereas viability was tested by the live/dead assay and analyzed by flow cytometry. The relative expression of interleukin-8 and monocyte chemoattractant protein-1 was investigated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Mitogen-activated protein kinase activation and mitogen-activated protein kinase phosphatase-1 expression were documented by Western blot analysis. We found that MMC induced corneal fibroblast elongation, apoptosis, and retarded cell growth, whereas DEX did not significantly alter cell morphology or viability. The combination of DEX and MMC did not induce additional apoptosis and cell death. DEX dose dependently down-regulated basal and MMC-induced interleukin-8 and monocyte chemoattractant protein-1 mRNA expression and protein secretion. DEX attenuated MMC-induced p38 and Jun N-terminal kinases activation and up-regulated expression. These suggested that DEX may inhibit MMC-induced interleukin-8 and monocyte chemoattractant protein-1 by up-regulating MKP-1 expression, which subsequently deactivated p38 and Jun N-terminal kinases activation. Combined MMC and DEX treatment may facilitate corneal wound healing.


Assuntos
Morte Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Córnea/citologia , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Glucocorticoides/farmacologia , Interleucina-8/metabolismo , Mitomicina/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córnea/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , MAP Quinase Quinase 4/metabolismo , Microscopia de Contraste de Fase , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Reação em Cadeia da Polimerase , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Cornea ; 27(6): 686-92, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18580261

RESUMO

PURPOSE: To study the morphologic changes and cytotoxicity in corneal fibroblasts after mitomycin C (MMC) treatment, ultraviolet B (UVB) irradiation, or in combination. METHODS: Primary porcine corneal fibroblasts, passages 3-5, were treated with MMC (0.1 or 0.2 mg/mL, ie, 0.01% or 0.02%, for 5 minutes), UVB irradiation (2, 5, or 10 mJ/cm2), or in combination. Control cells were treated with culture medium as a sham procedure. Alterations in cell morphology were documented by phase-contrast microscopy; cellular apoptosis was evaluated by Annexin V/propidium iodide staining and analyzed by flow cytometry; cytotoxicity was evaluated by lactate dehydrogenase assay; and cell growth was studied by genomic DNA quantification with the PicoGreen assay. RESULTS: UV irradiation induced significant dose-dependent corneal fibroblast rounding and detachment and cytotoxicity. MMC at 0.1 or 0.2 mg/mL induced considerable cell elongation and retarded cell proliferation at similar rates. MMC treatment alone did not cause significant apoptosis or cytotoxicity. However, MMC treatment before UV irradiation potentiated UV-related cytotoxicity proportional to the UV radiation dose. CONCLUSIONS: UV irradiation causes dose-dependent cytotoxicity in porcine corneal fibroblasts. MMC pretreatment potentiates UV-related cytotoxicity.


Assuntos
Alquilantes/farmacologia , Córnea/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Mitomicina/farmacologia , Raios Ultravioleta , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Terapia Combinada , Relação Dose-Resposta à Radiação , Sinergismo Farmacológico , Fibroblastos/enzimologia , Citometria de Fluxo , L-Lactato Desidrogenase/metabolismo , Microscopia de Contraste de Fase , Suínos
10.
J Refract Surg ; 24(2): 150-9, 2008 02.
Artigo em Inglês | MEDLINE | ID: mdl-18297939

RESUMO

PURPOSE: To explore inflammation and wound healing in the rabbit eye following topical ethanol treatment or mechanical debridement. METHODS: Seventy-six pigmented rabbit corneas were divided into four groups: mechanical group (n = 33), which received mechanical epithelial debridement; ethanol-30 (n = 18) and ethanol-60 groups (n = 18), which were treated with 20% ethanol for 30 and 60 seconds, respectively; and control group (n = 7), which remained untreated. Corneal epithelial and stromal keratocyte changes were examined with hematoxylin-eosin and terminal deoxynucleotidyltransferase-mediated dUPT nick end labeling (TUNEL) staining at 3 hours (day 0) and days 1, 2, 3, 5, and 7. Interleukin (IL)-1alpha, IL-8, monocyte chemotactic protein (MCP-1), and transforming growth factor (TGF)-beta1 expression were examined using real-time polymerase chain reaction. RESULTS: Stromal keratocyte cell death was higher in the mechanical group on day 0 (P = .002) and in the ethanol-60 group on days 3, 5, and 7 (P < .05). Keratocyte cell death was more pronounced in the ethanol-60 group than in the ethanol-30 group. In the mechanical group, IL-1alpha, IL-8, and MCP-1 expression was up-regulated starting on day 0 (P < .05) and returned to baseline on day 5 to 7. TGF-beta1 expression was up-regulated in the mechanical group throughout the experiment (P < .05). In the ethanol-30 and ethanol-60 groups, IL-1alpha expression was up-regulated on day 0, IL-8 expression was slightly up-regulated on day 0, and MCP-1 and TGF-beta1 expression were not up-regulated. CONCLUSIONS: Mechanical epithelial removal initially induces more keratocyte cell death, but deep stromal keratocyte death persists longer with ethanol treatment. In this rabbit model, mechanical epithelial removal upregulated inflammatory cytokines and TGF-beta1 gene expression more than ethanol treatment.


Assuntos
Citocinas/genética , Desbridamento/métodos , Epitélio Anterior/efeitos dos fármacos , Epitélio Anterior/cirurgia , Etanol/administração & dosagem , Regulação da Expressão Gênica/fisiologia , Animais , Morte Celular , Quimiocina CCL2/genética , Substância Própria/patologia , Epitélio Anterior/metabolismo , Marcação In Situ das Extremidades Cortadas , Interleucina-1alfa/genética , Interleucina-8/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/genética , Regulação para Cima , Cicatrização
11.
Invest Ophthalmol Vis Sci ; 48(5): 2009-16, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17460254

RESUMO

PURPOSE: To investigate the expression of chemokines and their signaling pathways after application of mitomycin C (MMC) to corneal fibroblasts. METHODS: Primary porcine and human corneal fibroblasts from passages 3 to 6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2 mg/mL for 1, 2, 5, or 10 minutes. The relative expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific antiphosphokinase antibodies. The signaling pathways by which MMC regulates the expression of IL-8 and MCP-1 were evaluated by pharmacological kinase-specific inhibitors. RESULTS: The expression of IL-8 and MCP-1 were upregulated after MMC treatment in a time- and concentration-dependent manner. Furthermore, the upregulated expression of IL-8 and MCP-1 increased with longer incubation time. MMC treatment enhanced the phosphorylation of p38, JNK, and ERK at different time points. The MMC-related IL-8 and MCP-1 expression was inhibited by both a p38 inhibitor (SB203580) and an ERK inhibitor (PD98059). A JNK inhibitor (SP600125) reduced the expression of MMC-induced MCP-1 but not of IL-8. CONCLUSIONS: MMC treatment upregulated the expression of IL-8 and MCP-1 mRNA and protein secretion by the activation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts.


Assuntos
Alquilantes/farmacologia , Quimiocina CCL2/metabolismo , Substância Própria/efeitos dos fármacos , Interleucina-8/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitomicina/farmacologia , Animais , Western Blotting , Células Cultivadas , Substância Própria/citologia , Substância Própria/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Fatores de Tempo , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Cornea ; 25(9): 1072-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17133057

RESUMO

PURPOSE: To explore the effect of ethanol treatment on corneal stromal cells. METHODS: Primary porcine corneal fibroblasts from passages 3 to 5 were treated with ethanol at concentrations of 10%, 15%, 20%, and 50% for 30 seconds. A control group was treated with phosphate-buffered saline (PBS) for 30 seconds. Morphologic changes were documented with phase-contrast microscopy, and the growth curves were examined with a PicoGreen assay. Cellular viability was examined with an ethidium homodimer and calcein-AM stain, whereas cellular apoptosis and/or necrosis were analyzed by a YO-PRO-1 dye/propidium iodide apoptosis assay coupled with flow cytometry and further confirmed with a genomic DNA pattern assay. Cellular toxicity was examined with a lactate dehydrogenase (LDH) assay. RESULTS: Significant cell rounding and detachment from the culture dish were noticed after 20% ethanol treatment of 30 seconds, despite that the cell morphology remained unchanged in the PBS and 10% and 15% ethanol groups. Twenty percent ethanol induced significant cellular toxicity, causing cell death as shown by ethidium homodimer and calcein-AM stain, YO-PRO-1 dye/propidium iodide apoptosis assay, and LDH assay, although 10% and 15% ethanol caused minimal changes to corneal fibroblasts. Cellular death was most significant 6 hours after the 20% ethanol treatment. The genomic DNA pattern revealed intact DNA in the control, 10% ethanol, and 15% ethanol groups at all times, whereas DNA smearing was noticed at 48 hours after the 20% ethanol treatment. However, none of the DNA examined revealed significant DNA laddering patterns of apoptosis. Fifty percent ethanol treatment of 30 seconds resulted in cell fixation and cell death. CONCLUSION: Treatment with 20% ethanol for 30 seconds induced significant porcine corneal fibroblast cell death, whereas 10% and 15% ethanol treatment of 30 seconds caused minimal changes. We propose that, when applied for 30 seconds, 20% ethanol is the threshold level that causes cell death in cultured porcine corneal fibroblasts.


Assuntos
Apoptose/efeitos dos fármacos , Substância Própria/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Etanol/toxicidade , Solventes/toxicidade , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Substância Própria/enzimologia , Substância Própria/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Citometria de Fluxo , L-Lactato Desidrogenase/metabolismo , Suínos
13.
J Lipid Res ; 45(11): 2116-22, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15314101

RESUMO

Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation. These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis.


Assuntos
Lipoproteína-X/metabolismo , Cirrose Hepática Biliar/metabolismo , Oxigênio/metabolismo , Animais , Aorta/citologia , Apoptose , Bovinos , Células Cultivadas , Cromatografia em Gel , Regulação para Baixo , Eletroforese em Gel de Ágar , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/metabolismo , História do Século XX , Ligantes , Lipoproteínas LDL/metabolismo , Peptídeos/química , RNA Mensageiro/metabolismo , Fatores de Tempo
14.
Eur J Biochem ; 270(8): 1855-62, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12694199

RESUMO

Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis-regulatory elements and transcription factors involved in this process in HL-60 cells. cis-Regulatory elements in the DNase II promoter were located by 5' deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Regulação Neoplásica da Expressão Gênica/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Sequência de Bases , Diferenciação Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Primers do DNA , Regulação Enzimológica da Expressão Gênica/genética , Células HL-60 , Humanos , Cinética , Luciferases/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Fator de Transcrição Sp3 , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Dedos de Zinco
15.
Biochem Biophys Res Commun ; 296(1): 48-53, 2002 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-12147225

RESUMO

Several recent studies have suggested that intracellular deoxyribonuclease II (DNase II) is responsible for the degradation of DNA from apoptotic cells that are engulfed by macrophages. In this study, we studied DNase II expression during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. Basal levels of DNase II mRNA and protein were low, with expression being up-regulated approximately 15- and 7-fold, respectively, in HL-60 and THP-1 cells 72 h after PMA treatment. Nuclear run-on and luciferase reporter assays showed that transcription of DNase II gene was increased in PMA-treated cells. Together, these results demonstrate that DNase II gene transcription is increased during myelomonocytic differentiation, resulting in increased levels of mRNA and protein. This increase in DNase II levels in differentiated HL-60 and THP-1 cells suggests that it may play an important role in macrophages.


Assuntos
Diferenciação Celular , Endodesoxirribonucleases/genética , Regulação Enzimológica da Expressão Gênica , Monócitos/citologia , Regulação para Cima , Sequência de Bases , Linhagem Celular , Primers do DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Células HL-60/enzimologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacos
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