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1.
PLoS One ; 12(6): e0178995, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28594934

RESUMO

Head and neck paragangliomas (HNPGLs) are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3ß/ß-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3ß/ß-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL.


Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , PPAR alfa/antagonistas & inibidores , PPAR alfa/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Caspase 6/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Caspases/metabolismo , Caspases Iniciadoras/metabolismo , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Imuno-Histoquímica , Oxazóis/farmacologia , PPAR alfa/agonistas , Pirimidinas/farmacologia , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/farmacologia , Cicatrização/efeitos dos fármacos
2.
Diabetes Obes Metab ; 19(2): 257-265, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27761989

RESUMO

AIMS: Diet-derived short chain fatty acids (SCFAs) improve glucose homeostasis in vivo, but the role of individual SCFAs and their mechanisms of action have not been defined. This study evaluated the effects of increasing colonic delivery of the SCFA propionate on ß-cell function in humans and the direct effects of propionate on isolated human islets in vitro. MATERIALS AND METHODS: For 24 weeks human subjects ingested an inulin-propionate ester that delivers propionate to the colon. Acute insulin, GLP-1 and non-esterified fatty acid (NEFA) levels were quantified pre- and post-supplementation in response to a mixed meal test. Expression of the SCFA receptor FFAR2 in human islets was determined by western blotting and immunohistochemistry. Dynamic insulin secretion from perifused human islets was quantified by radioimmunoassay and islet apoptosis was determined by quantification of caspase 3/7 activities. RESULTS: Colonic propionate delivery in vivo was associated with improved ß-cell function with increased insulin secretion that was independent of changes in GLP-1 levels. Human islet ß-cells expressed FFAR2 and propionate potentiated dynamic glucose-stimulated insulin secretion in vitro, an effect that was dependent on signalling via protein kinase C. Propionate also protected human islets from apoptosis induced by the NEFA sodium palmitate and inflammatory cytokines. CONCLUSIONS: Our results indicate that propionate has beneficial effects on ß-cell function in vivo, and in vitro analyses demonstrated that it has direct effects to potentiate glucose-stimulated insulin release and maintain ß-cell mass through inhibition of apoptosis. These observations support ingestion of propiogenic dietary fibres to maintain healthy glucose homeostasis.


Assuntos
Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Propionatos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Adulto , Idoso , Western Blotting , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Colo , Gorduras na Dieta , Ésteres/farmacologia , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos Voláteis , Feminino , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Inulina/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo
3.
Asian J Androl ; 19(1): 47-51, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-26643564

RESUMO

Recent studies have reported that caspase 7 has an apoptotic and nonapoptotic function. However, the relationship between caspase 7 and spermatogenesis remains unknown. This study aimed to investigate the possible function of caspase 7 during normal and abnormal spermatogenesis. The cleaved form of caspase 7 was detected in testis tissues at different postpartum times (5-14 weeks) by qRT-PCR, Western blot and immunohistochemistry (IHC). Then, the mice models of spermatogenic dysfunction were obtained by busulfan (30 mg kg-1 to further evaluate the potential function and mechanism of caspase 7. qRT-PCR and Western blot results showed that caspase 7 expression was gradually elevated from 5 to 14 weeks, which was not connected with apoptosis. IHC results revealed that caspase 7 was mainly located in spermatogenic cells and Leydig cells. In addition, spermatogenic dysfunction induced by busulfan gradually enhanced the apoptosis and elevated the expression of caspase 3, caspase 6, and caspase 9, but decreased the expression of caspase 7 in spermatogenic cells. However, when spermatogenic cells were mostly disappeared at the fourth week after busulfan treatment, caspase 7 expression in Leydig cells was significantly increased and positively correlated with the expression of caspase 3, caspase 6, and caspase 9. Therefore, these results indicate that caspase 7 has a nonapoptic function that participates in normal spermatogenesis, but also displays apoptotic function in spermatogenic dysfunction.


Assuntos
Apoptose/genética , Caspase 7/genética , Células Intersticiais do Testículo/metabolismo , Espermatogênese/genética , Alquilantes/toxicidade , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Bussulfano/toxicidade , Caspase 3/efeitos dos fármacos , Caspase 3/genética , Caspase 6/efeitos dos fármacos , Caspase 6/genética , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 9/efeitos dos fármacos , Caspase 9/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogênese/efeitos dos fármacos , Testículo/metabolismo
4.
Br J Cancer ; 114(2): 177-87, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26695443

RESUMO

BACKGROUND: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. METHODS: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. RESULTS: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells. CONCLUSIONS: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Interferon gama/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Fator de Transcrição STAT1/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos/efeitos dos fármacos , Antígenos/genética , Antígenos/metabolismo , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Capecitabina/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/genética , Caspase 7/metabolismo , Cisplatino/farmacologia , Citocinas/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Proteínas do Citoesqueleto/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Interferon beta/efeitos dos fármacos , Interferon beta/genética , Interferon beta/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Resistência a Myxovirus/efeitos dos fármacos , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Transplante de Neoplasias
5.
Dis Esophagus ; 29(6): 656-62, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25894100

RESUMO

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers in Japan. Anticancer chemotherapy has been useful for ESCC treatment. However, therapeutic options are limited. Recently, bisphosphonates (BPs), which are osteoporosis drugs, have shown anticancer effects in several cancer cell lines, but the effects against ESCC cell lines are unknown. In this study, we examined the cytotoxic effects of BPs and their mechanisms of cytotoxicity in human ESCC cell lines. A first-generation BP (etidronate), two second-generation BPs (alendronate and pamidronate), and two third-generation BPs (risedronate and zoledronate) were used in this study. All BPs, except etidronate, were cytotoxic, as indicated by increased caspase-3/7 activity and numbers of Annexin-fluorescein isothiocyanate positive cells in ESCC cell lines. From cell cycle analysis, G0/G1-phase arrest was observed upon treatment with second- and third-generation BPs. In addition, Cyclin D1 protein expression levels were decreased by second- and third-generation BP treatment. Although squalene and trans, trans-farnesol minimally affected BP cytotoxicity, treatment with geranylgeraniol inhibited BP cytotoxicity almost completely. We concluded that second- and third-generation BPs are cytotoxic to ESCC cell lines as they induce apoptosis and inhibit the cell cycle through mevalonate pathway inhibition. Therefore, BP treatment may be a beneficial therapy in ESCC patients.


Assuntos
Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Carcinoma de Células Escamosas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Difosfonatos/farmacologia , Neoplasias Esofágicas/patologia , Anexinas/efeitos dos fármacos , Anexinas/metabolismo , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/efeitos dos fármacos , Ciclina D1/metabolismo , Diterpenos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Carcinoma de Células Escamosas do Esôfago , Farneseno Álcool/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Esqualeno/farmacologia
6.
Braz J Med Biol Res ; 48(2): 111-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25517919

RESUMO

Pancreatic cancer is the fourth leading cause of cancer death. Gemcitabine is widely used as a chemotherapeutic agent for the treatment of pancreatic cancer, but the prognosis is still poor. Berberine, an isoquinoline alkaloid extracted from a variety of natural herbs, possesses a variety of pharmacological properties including anticancer effects. In this study, we investigated the anticancer effects of berberine and compared its use with that of gemcitabine in the pancreatic cancer cell lines PANC-1 and MIA-PaCa2. Berberine inhibited cell growth in a dose-dependent manner by inducing cell cycle arrest and apoptosis. After berberine treatment, the G1 phase of PANC-1 cells increased by 10% compared to control cells, and the G1 phase of MIA-PaCa2 cells was increased by 2%. Whereas gemcitabine exerts antiproliferation effects through S-phase arrest, our results showed that berberine inhibited proliferation by inducing G1-phase arrest. Berberine-induced apoptosis of PANC-1 and MIA-PaCa2 cells increased by 7 and 2% compared to control cells, respectively. Notably, berberine had a greater apoptotic effect in PANC-1 cells than gemcitabine. Upon treatment of PANC-1 and MIA-PaCa2 with berberine at a half-maximal inhibitory concentration (IC50), apoptosis was induced by a mechanism that involved the production of reactive oxygen species (ROS) rather than caspase 3/7 activation. Our findings showed that berberine had anti-cancer effects and may be an effective drug for pancreatic cancer chemotherapy.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Berberina/uso terapêutico , Fase G1/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Caspase 3/efeitos dos fármacos , Caspase 7/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Citometria de Fluxo , Humanos , Fatores de Tempo , Gencitabina
7.
J Dent Res ; 93(4): 394-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24492932

RESUMO

Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts.


Assuntos
Interleucina-6/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Células 3T3 , Fosfatase Alcalina/efeitos dos fármacos , Animais , Densidade Óssea/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 7/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Meios de Cultivo Condicionados , Regulação da Expressão Gênica/genética , Antígeno Ki-67/efeitos dos fármacos , Camundongos , Osteocalcina/efeitos dos fármacos , Fluxo Pulsátil/fisiologia , Ligante RANK/efeitos dos fármacos , Estresse Mecânico
8.
Int J Oncol ; 43(2): 622-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23732481

RESUMO

Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 9/efeitos dos fármacos , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Leucemia/tratamento farmacológico , Camundongos , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Células U937
9.
Parasitol Int ; 62(2): 100-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23159324

RESUMO

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.


Assuntos
Apoptose/fisiologia , Proteínas de Helminto/metabolismo , Estágios do Ciclo de Vida , Schistosoma japonicum/crescimento & desenvolvimento , Esquistossomose Japônica/parasitologia , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Animais , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Inibidores de Caspase/farmacologia , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica , Proteínas de Helminto/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Schistosoma japonicum/citologia , Schistosoma japonicum/efeitos dos fármacos , Schistosoma japonicum/fisiologia , Alinhamento de Sequência , Transdução de Sinais , Organismos Livres de Patógenos Específicos
10.
Biomacromolecules ; 14(2): 367-76, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23210662

RESUMO

We describe the synthesis of a core-shell biohybrid consisting of a human serum albumin (HSA) core that serves as a reservoir for lipophilic molecules and a cationized shell region consisting of ethynyl-G2.0-PAMAM or ethynyl-G3.0-PAMAM dendrons. The binding capacity of lipophilic guests was quantified applying electron paramagnetic resonance (EPR) spectroscopy, and five to six out of seven pockets were still available compared with HSA. The attachment of ethynyl-G2.0-PAMAM dendrons to HSA yielded a nontoxic core-shell macromolecule that was clearly uptaken by A549 human epithelial cells due to the presence of the dendritic PAMAM shell. Significantly higher loading of doxorubicin was observed for dendronized G2-DHSA compared with the native protein due to the availability of binding pockets of the HSA core, and interaction with the dendritic shell. Dendronized G2-DHSA-doxorubicin displayed significant cytotoxicity resulting from high drug loading and high stability under different conditions, thus demonstrating its great potential as a transporter for drug molecules.


Assuntos
Dendrímeros/química , Dendrímeros/metabolismo , Portadores de Fármacos/farmacologia , Albumina Sérica/farmacologia , Carcinoma/tratamento farmacológico , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Células Epiteliais/metabolismo , Humanos , Albumina Sérica/química
11.
J Nat Prod ; 75(5): 865-9, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22530779

RESUMO

Activity-guided fractionation of a petroleum ether-soluble extract of the roots of Onosma paniculata, which has been shown to affect the cell cycle and to induce apoptosis in melanoma cells, led to the isolation of several shikonin derivatives, namely, ß-hydroxyisovalerylshikonin (1), acetylshikonin (2), dimethylacrylshikonin (3), and a mixture of α-methylbutyrylshikonin and isovalerylshikonin (4+5). All compounds exhibited strong cytotoxicity against eight cancer cell lines and MRC-5 lung fibroblasts, with 3 found to possess the most potent cytotoxicity toward four melanoma cell lines (SBcl2, WM35, WM9, and WM164). Furthermore, 3 and the mixture of 4+5 were found to interfere with cell-cycle progression in these cell lines and led to an increasing number of cells in the subG1 region as well as to caspase-3/7 activation, indicating apoptotic cell death.


Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Boraginaceae/química , Ciclo Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Naftoquinonas/isolamento & purificação , Naftoquinonas/farmacologia , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/química , Humanos , Melanoma , Estrutura Molecular , Naftoquinonas/química , Raízes de Plantas/química
12.
Int Endod J ; 45(5): 401-12, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22142405

RESUMO

AIM: To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs). METHODOLOGY: Dental pulp stem cells were exposed to 5, 10, 20 or 40 µmol L(-1) of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student's t-test. RESULTS: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families - extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9. CONCLUSION: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.


Assuntos
Cisplatino/toxicidade , Citostáticos/toxicidade , Polpa Dentária/citologia , Ectoderma/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 7/efeitos dos fármacos , Caspase 8/efeitos dos fármacos , Caspase 9/efeitos dos fármacos , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Ectoderma/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutagênicos/toxicidade , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Serina/efeitos dos fármacos , Pele/citologia , Pele/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos
13.
Toxicol Appl Pharmacol ; 251(3): 217-25, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21237187

RESUMO

Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC50 (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC50 (concentration at which 50% of maximal cell death is inhibited) of 0.6 µM and 0.4 µM, respectively. In contrast, MTHF (up to 10 µM) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.


Assuntos
Homocisteína/toxicidade , Neurônios Motores/efeitos dos fármacos , Tetra-Hidrofolatos/farmacologia , Vitamina B 12/análogos & derivados , Complexo Vitamínico B/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Homocisteína/administração & dosagem , Concentração Inibidora 50 , Camundongos , Neurônios Motores/metabolismo , Neuroblastoma/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tetra-Hidrofolatos/administração & dosagem , Fatores de Tempo , Vitamina B 12/administração & dosagem , Vitamina B 12/farmacologia , Complexo Vitamínico B/administração & dosagem
14.
J Clin Periodontol ; 38(3): 208-18, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21214612

RESUMO

AIMS: To determine the effect of nicotine, cotinine and cigarette smoke extract (CSE) on the neutrophil respiratory burst and their effect on activation of the nuclear factor-κB (NFκB) pathway in oral epithelium. MATERIALS AND METHODS: Neutrophils from periodontally healthy individuals were treated with nicotine, cotinine and CSE before stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli lipopolysaccharide. Total and extracellular reactive oxygen species (ROS) generation was determined by luminol/isoluminol chemiluminescence. Activation of NFκB in oral epithelial cells was determined by immunocytochemistry. RESULTS: Smoke extract alone caused increased neutrophil extracellular isoluminol-dependent chemiluminescence, not detectable with luminol. However, pre-treatment with smoke extract reduced both total and extracellular ROS generation in response to all stimuli. Nicotine and cotinine had no effect on the neutrophil respiratory burst. Smoke extract, nicotine and cotinine did not induce oral epithelial cell NFκB activation. CONCLUSIONS: These data demonstrate that smoke extract reduces the ability of neutrophils to generate ROS after stimulation with F. nucleatum and IgG-opsonized S. aureus but, at high concentrations, stimulates extracellular ROS generation. During periodontitis, cigarette smoking may differentially affect neutrophil function, generally preventing elimination of periodontal pathogens but, in heavy smokers, also stimulating ROS release and oxidative stress mediated tissue damage.


Assuntos
Cotinina/farmacologia , Neutrófilos/efeitos dos fármacos , Nicotiana , Nicotina/farmacologia , Explosão Respiratória/efeitos dos fármacos , Fumaça , Anticorpos Antibacterianos/imunologia , Caspase 3/efeitos dos fármacos , Caspase 7/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Escherichia coli , Fusobacterium nucleatum/imunologia , Humanos , Imunoglobulina G/imunologia , Imuno-Histoquímica , Lipopolissacarídeos/farmacologia , Substâncias Luminescentes , Medições Luminescentes , Luminol/análogos & derivados , Mucosa Bucal/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/imunologia
15.
Cancer Chemother Pharmacol ; 65(1): 143-50, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19449007

RESUMO

PURPOSE: We tried to clarify the cytotoxic mechanism of VK(3) using the breast cancer cell line MCF-7. METHODS: Cytotoxicity was measured via intracellular esterase activity. DNA fragmentation was assessed by agarose gel electrophoresis. JC-1 staining was applied to measure mitochondrial dysfunction. Caspase activation and reactive oxidative species (ROS) generation were also measured. RESULTS: VK(3) exhibited cytotoxicity that caused DNA fragmentation in MCF-7 cells with an IC(50) of 14.2 microM. JC-1 staining revealed that VK(3) caused mitochondrial dysfunction including a disappearance of mitochondrial membrane potential. Additional investigation showed that the mitochondrial damage was induced by the generation of ROS and the subsequent activation of caspase-7 and -9. CONCLUSIONS: Our findings demonstrate that VK(3)-induced apoptosis is selectively initiated by the mitochondria-related pathway and might be useful in breast cancer chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Vitamina K 3/farmacologia , Antineoplásicos/administração & dosagem , Neoplasias da Mama/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 9/efeitos dos fármacos , Caspase 9/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Ágar , Feminino , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Vitamina K 3/administração & dosagem
16.
Toxicology ; 259(1-2): 69-76, 2009 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-19428945

RESUMO

The purpose of the current study is to understand the effects of nicotine in human retinal pigment epithelial (ARPE-19), human microvascular endothelial cells (HMVEC) and rat neurosensory retinal (R28) cells. ARPE-19, HMVEC and R28 cell cultures were treated with 10(-2) and 10(-4)M nicotine for 24h. R28 cells were also pre-treated for 4h with ALLN and ALLM (calpain inhibitors) or epicatechin, an antioxidant flavonoid compound. Trypan blue dye exclusion assay, caspase-3/7, LDH activity and DNA laddering assays were performed. With 10(-2)M nicotine treatment, R28 cell cultures showed decreased cell viability that was partially reversed by pre-treatment with the antioxidant epicatechin but not with calpain inhibitors. The DNA ladder assay showed a 200bp banding pattern consistent with apoptosis, however, caspase-3/7 activity was not increased. After treatment with 10(-2)M nicotine, HMVEC cultures showed decreased cell viability and increased LDH activity but no DNA banding patterns or caspase-3/7 activity. ARPE-19 cells showed no change in cell viability or caspase-3/7 activity at any of the nicotine concentrations. We conclude of dissimilar responses to nicotine treatment in three different cell lines. Nicotine was toxic to HMVEC and R28 cell cultures but the ARPE-19 cells were unaffected. In R28 cells, the nicotine effects were through an oxidant pathway that is non-caspase, non-calpain mediated while the HMVEC toxicity was via necrosis. Understanding the mechanisms of cell death may have potential therapeutic implications in the treatment of cigarette smoking related retinal diseases such as age-related macular degeneration (AMD).


Assuntos
Células Endoteliais/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Catequina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Necrose/induzido quimicamente , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Ratos , Retina/citologia , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo
17.
Toxicol In Vitro ; 23(5): 834-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19406223

RESUMO

The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.


Assuntos
Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Flavonoides/farmacologia , Fenóis/farmacologia , Chá/química , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Catequina/isolamento & purificação , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/isolamento & purificação , Humanos , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fenóis/isolamento & purificação , Polifenóis , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
18.
Arch Toxicol ; 83(6): 601-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19165469

RESUMO

This study is the first to examine the increased apoptosis in the adult rat ovary after lactational exposure to coumestrol (COU), a potent phytoestrogen. Lactating dams were gavaged at doses of 0.01, 0.1, 1, and 10 mg/kg COU during the lactation period and the reproductive effects of female pups were investigated in young adults. Rats were sacrificed at postnatal days (PND) 81-84. Ovarian weights were reduced significantly at 0.1 and 1.0 mg/kg COU. The reduction in the ovarian weight occurred in parallel with an increase in the apoptosis at PND 135-140. A marked dose-dependent increase in the expressions of active caspase-3 and -7 was observed in ovarian granulosa cells. Immunostaining for active caspase-3 and the TUNEL staining of apoptotic cells were also increased in ovaries exposed to COU in a dose-dependent manner. These results suggest new sights into the effect of lactational exposure to COU on the female reproductive health.


Assuntos
Apoptose/efeitos dos fármacos , Cumestrol/toxicidade , Ovário/efeitos dos fármacos , Fitoestrógenos/toxicidade , Animais , Animais Recém-Nascidos , Animais Lactentes , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Cumestrol/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Marcação In Situ das Extremidades Cortadas , Tamanho do Órgão/efeitos dos fármacos , Ovário/citologia , Fitoestrógenos/administração & dosagem , Gravidez , Ratos , Ratos Sprague-Dawley , Reprodução/efeitos dos fármacos
19.
Toxicology ; 259(1-2): 1-9, 2009 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-19167454

RESUMO

There are increasing concerns regarding the relative safety of chlorpyrifos (CPF) to various facets of the environment. Although published works suggest that CPF is relatively safe in adult animals, recent evidence indicates that juveniles, both animals and humans, may be more sensitive to CPF toxicity than adults. In young animals, CPF is neurotoxic and mechanistically interferes with cellular replication and cellular differentiation, which culminates in the alteration of synaptic neurotransmission in neurons. However, the effects of CPF on glial cells are not fully elucidated. Here we report that chlorpyrifos is toxic to oligodendrocyte progenitors. In addition, CPF produced dose-dependent increases in 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) and dihydroethidium (DHE) fluorescence intensities relative to the vehicle control. Moreover, CPF toxicity is associated with nuclear condensation and elevation of caspase 3/7 activity and Heme oxygenase-1 mRNA expression. Pan-caspase inhibitor QVDOPh and cholinergic receptor antagonists' atropine and mecamylamine failed to protect oligodendrocyte progenitors from CPF-induced injury. Finally, glutathione (GSH) depletion enhanced CPF-induced toxicity whereas nitric oxide synthetase inhibitor L-NAME partially protected progenitors and the non-specific antioxidant vitamin E (alpha-tocopherol) completely spared cells from injury. Collectively, this data suggests that CPF induced toxicity is independent of cholinergic stimulation and is most likely caused by the induction of oxidative stress.


Assuntos
Clorpirifos/toxicidade , Inseticidas/toxicidade , Oligodendroglia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Clorpirifos/administração & dosagem , Relação Dose-Resposta a Droga , Etídio/análogos & derivados , Etídio/metabolismo , Fluoresceínas/metabolismo , Fluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/efeitos dos fármacos , Heme Oxigenase-1/genética , Inseticidas/administração & dosagem , Oligodendroglia/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Células-Tronco/metabolismo
20.
J Pharmacol Exp Ther ; 328(3): 708-14, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19074681

RESUMO

The platinum compound cisplatin is one of the most potent chemotherapy agents available to treat various malignancies. Nephrotoxicity is a common complication of cisplatin chemotherapy, which involves increased oxidative and nitrosative stress, limiting its clinical use. In this study, we have investigated the effects of a nonpsychoactive cannabinoid cannabidiol, which was reported to exert antioxidant effects and has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in patients in a mouse model of cisplatin-induced nephropathy. Cisplatin induced increased expression of superoxide-generating enzymes RENOX (NOX4) and NOX1, enhanced reactive oxygen species generation, inducible nitric-oxide synthase expression, nitrotyrosine formation, apoptosis (caspase-3/7 activity, DNA fragmentation, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining), poly(ADP-ribose) polymerase activity, and inflammation (tumor necrosis factor-alpha and interleukin-1beta) in the kidneys of mice, associated with marked histopathological damage and impaired renal function (elevated serum blood urea nitrogen and creatinine levels) 72 h after the administration of the drug. Treatment of mice with cannabidiol markedly attenuated the cisplatin-induced oxidative/nitrosative stress, inflammation, and cell death in the kidney, and it improved renal function. Thus, our results suggest that cannabidiol may represent a promising new protective strategy against cisplatin-induced nephrotoxicity.


Assuntos
Canabidiol/farmacologia , Morte Celular/efeitos dos fármacos , Cisplatino/toxicidade , Inflamação/prevenção & controle , Rim/patologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Cisplatino/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/genética
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