RESUMO
Although hepatitis C virus (HCV) affects approximately 130-170 million people worldwide, no vaccines are available. HCV is an important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, leading to the need for liver transplantation. In this study, curcumin, a constituent used in traditional Chinese medicine, has been evaluated for its anti-HCV activity and mechanism, using a human hepatoma cell line containing the HCV genotype 1b subgenomic replicon. Below the concentration of 20% cytotoxicity, curcumin dose-dependently inhibited HCV replication by luciferase reporter gene assay, HCV RNA detection and HCV protein analysis. Under the same conditions, curcumin also dose-dependently induced heme oxygenase-1 with the highest induction at 24 h. Hemin, a heme oxygenase-1 inducer, also inhibited HCV protein expression in a dose-dependent manner. The knockdown of heme oxygenase-1 partially reversed the curcumin-inhibited HCV protein expression. In addition to the heme oxygenase-1 induction, signaling molecule activities of AKT, extracellular signal-regulated kinases (ERK) and nuclear factor-κB (NF-κB) were inhibited by curcumin. Using specific inhibitors of PI3K-AKT, MEK-ERK and NF-κB, the results suggested that only PI3K-AKT inhibition is positively involved in curcumin-inhibited HCV replication. Inhibition of ERK and NF-κB was likely to promote HCV protein expression. In summary, curcumin inhibited HCV replication by heme oxygenase-1 induction and AKT pathway inhibition. Although curcumin also inhibits ERK and NF-κB activities, it slightly increased the HCV protein expression. This result may provide information when curcumin is used as an adjuvant in anti-HCV therapy.
Assuntos
Antivirais/farmacologia , Curcumina/farmacologia , Heme Oxigenase-1/metabolismo , Hepacivirus/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Replicação Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/genética , Hemina/farmacologia , Hemina/fisiologia , Humanos , Interferência de RNA , RNA Viral/biossíntese , RNA Viral/genética , Transdução de Sinais , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genéticaRESUMO
PURPOSE: Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino. The authors investigated the effects of Gyp on cell morphology, viability, cell cycle distribution, and induction of apoptosis in human oral cancer SAS cells and the determination of murine SAS xenograft model in vivo. EXPERIMENTAL DESIGN: Flow cytometry was used to quantify the percentage of viable cells; cell cycle distribution; sub-G1 phase (apoptosis); caspase-3, -8, and -9 activity; reactive oxygen species (ROS) production, intracellular Ca(2+) determination; and the level of mitochondrial membrane potential (ΔΨ(m)). Western blotting was used to examine levels of apoptosis-associated proteins, and confocal laser microscopy was used to examine the translocation of proteins in cells. RESULTS: Gyp induced morphological changes, decreased the percentage of viable cells, caused G0/G1 phase arrest, and triggered apoptotic cell death in SAS cells. Cell cycle arrest induced by Gyp was associated with apoptosis. The production of ROS, increased intracellular Ca(2+) levels, and the depolarization of ΔΨ(m) were observed. Gyp increased levels of the proapoptotic protein Bax but inhibited the levels of the antiapoptotic proteins Bcl-2 and Bcl-xl. Gyp also stimulated the release of cytochrome c and Endo G. Translocation of GADD153 to the nucleus was stimulated by Gyp. Gyp in vivo attenuated the size and volume of solid tumors in a murine xenograft model of oral cancer. CONCLUSIONS: Gyp-induced cell death occurs through caspase-dependent and caspase-independent apoptotic signaling pathways, and the compound reduced tumor size in a xenograft nu/nu mouse model of oral cancer.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias Bucais/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Fase G1/efeitos dos fármacos , Gynostemma/química , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Extratos Vegetais/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Gallic acid (GA) induces apoptosis in different types of cancer cell lines. In this study, we investigate the apoptotic effects induced by GA in human promyelocytic leukemia HL-60 cells, and clarify the underlying mechanism. Our results showed that GA reduced the viability of HL-60 cells in a dose- and time-dependent manner. GA led to G(0)/G(1) phase arrest in HL-60 cells through promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. GA caused DNA damage and fragmentation in HL-60 cells as assayed using DAPI staining and Comet assay. Flow cytometric analysis revealed that GA increased Ca(2+) levels and reduced the mitochondrial membrane potential (ΔΨ(m)) in HL-60 cells. Apoptotic protein expressions were determined by Western blotting. The results indicated that GA-mediated apoptosis of HL-60 cells mainly depended on mitochondrial pathway, by promoting the release of cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) and by up-regulating the protein expression of Bcl-2-associated X protein (BAX), caspase-4, caspase-9 and caspase-3. In addition, GA also activated the death receptor-dependent pathway by enhancing the protein expressions of fatty acid synthase (FAS), FAS ligand (FASL), caspase-8 and BCL-2 interacting domain (BID). We determined the mRNA expression of the gene levels of these proteins by real-time PCR. The results showed that GA-mediated apoptosis of HL-60 cells mainly depended on up-regulation of the mRNA of caspase-8, caspase-9, caspase-3, AIF and Endo G. In conclusion, GA-induced apoptosis occurs through the death receptor and mitochondria-mediated pathways. The evaluation of GA as a potential therapeutic agent for treatment of leukemia seems warranted.
Assuntos
Apoptose/efeitos dos fármacos , Ciclina D/antagonistas & inibidores , Ciclina E/antagonistas & inibidores , Fase G1/efeitos dos fármacos , Ácido Gálico/farmacologia , Mitocôndrias/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Sequência de Bases , Ensaio Cometa , Primers do DNA , Citometria de Fluxo , Células HL-60 , Humanos , Reação em Cadeia da PolimeraseRESUMO
Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, has been used as a folk medicine in the Chinese population for centuries and is known to have diverse pharmacologic effects, including anti-proliferative and anti-cancer actions. However, the effects of Gyp on prevention from invasion and migration of oral cancer cells are still unsatisfactory. The purpose of this study was to investigate effects of Gyp treatment on migration and invasion of SAS human oral cancer cells. SAS cells were cultured in the presence of 90 and 180 µg/mL Gyp for 24 and 48 hours. Gyp induced cytotoxic effects and inhibited SAS cells migration and invasion in dose- and time-dependent response. Wound-healing assay and boyden chamber assay were carried out to investigate Gyp-inhibited migration and invasion of SAS cells. Gyp decreased the abundance of several proteins, including nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/ 2), matrix metalloproteinase-9, -2 (MMP-9, -2), sevenless homolog (SOS), Ras, urokinase-type plasminogen activator (uPA), focal adhesion kinase (FAK) and RAC-alpha serine/threonine-protein kinase (Akt), in a time-dependent manner. In addition, Gyp decreased mRNA levels of MMP-2, MMP-7, MMP-9 but did not affect FAK and Rho A mRNA levels in SAS cells. These results provide evidences for the role of Gyp as a potent anti-metastatic agent, which can markedly inhibit the metastatic and invasive capacity of oral cancer cells. The inhibition of NF-κB and MMP-2, -7 and -9 signaling may be one of the mechanisms that is present in Gyp-inhibited cancer cell invasion and migration.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Metaloproteinases de Matriz , NF-kappa B/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gynostemma , Humanos , Metaloproteinase 2 da Matriz , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Bucais/enzimologia , Neoplasias Bucais/patologia , Invasividade Neoplásica , Extratos Vegetais/farmacologia , Transdução de Sinais , Fatores de TempoRESUMO
Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Neoplasias Bucais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Gynostemma , Humanos , Técnicas In Vitro , Medicina Tradicional Chinesa/métodos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Extratos Vegetais/farmacologiaRESUMO
AIM OF THE STUDY: Gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in Asia. Our previous study demonstrates that Gynostemma pentaphyllum n-butanol extract inhibits the onset and improves the recovery of CCl(4)-induced liver fibrogenesis in rats and inhibits PDGF-induced rat hepatic stellate cells (HSCs) proliferation. In this study, the effect of Gynostemma pentaphyllum extract on cytokines and type I procollagen expression was analyzed. MATERIALS AND METHODS: Rat HSCs were treated with PDGF, Gynostemma pentaphyllum n-butanol extract, RP-18-Gyp fraction, rapamycin or vehicle. Rat cytokine antibody array chip or ELISA kit was used for cytokines detection. Intracellular protein expression was detected by Western blotting, mRNA expression was analyzed by RT-PCR. RESULTS: RP-18-Gyp fraction is the more purified gypenosides fraction from Gynostemma pentaphyllum n-butanol extract. In cell proliferation, the inhibitory effect of 200 microg/ml RP-18-Gyp fraction is similar to 500 microg/ml Gynostemma pentaphyllum n-butanol extract. Furthermore, both of them have the ability of decreasing monocyte chemoattractant protein-1 (MCP-1) mRNA expression and protein release and inhibiting type I procollagen protein expression. CONCLUSIONS: Both of Gynostemma pentaphyllum n-butanol extract and its more purified RP-18-Gyp fraction have the biological activities in the inhibition of cell proliferation, MCP-1 release and type I procollagen expression in rat HSCs. These data could provide the evidence to support for the traditional use of Gynostemma pentaphyllum in hepatitis.
Assuntos
Quimiocina CCL2/metabolismo , Colágeno Tipo I/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Expressão Gênica/efeitos dos fármacos , Gynostemma/química , Células Estreladas do Fígado/metabolismo , Masculino , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Capsaicin, a pungent ingredient of red pepper, has been reported to possess antitumor activities. In this study, the effects of capsaicin on human HepG2 cells were investigated. Capsaicin reduced viability by PI incorporation in HepG2 cells in a dose and time dependent manner. Capsaicin promoted intracellular Ca2+ production and reactive oxygen species (ROS). The alpha psi(m) significantly decreased after capsaicin treatment for 24 h. Co-treatment of HepG2 cells with capsaicin and BAPTA (an intracellular Ca2+ chelator) significantly reduced intracellular Ca2+ levels, prevented alpha psi(m) disruption and inhibited apoptosis induction. The protein levels of Bcl-2 decreased and Bax increased in the mitochondrial fraction while the Bax protein decreased, and p53 and cytochrome c protein levels increased in the cytosolic fraction in HepG2 cells after capsaicin treatment for 24 h by Western blot. Immunostaining and confocal microscopic analysis also showed that capsaicin promoted cytoplasmic GADD153 expression and GRP78 nuclear translocation. The caspase-3 activity significantly increased after capsaicin treatment for 24 h. Our results indicated that the capsaicin-induced apoptosis in HepG2 cells may result from the elevation of intracellular Ca2+ production, ROS, disruption of alpha psi(m), regulation of Bcl-2 family protein expression and caspase-3 activity.
Assuntos
Apoptose/efeitos dos fármacos , Capsaicina/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/biossíntese , Chaperona BiP do Retículo Endoplasmático , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Espécies Reativas de Oxigênio/metabolismoRESUMO
Gypenosides (Gyp), a component of Gynostemma pentaphyllum Makino, was selected for examining the effects on the cell viability, cell cycle and induction of apoptosis in human tongue cancer SCC-4 cells. Gyp induced cytotoxicity (decreased the percentage of viable cells) in SCC-4 cells appeared to be associated with induction of cell cycle arrest (G0/G1 arrest), apoptotic cell death based on Gyp induced morphological changes and DNA fragmentation and increased the sub-G1 group in examined SCC-4 cells. The production of reactive oxygen species and Ca(2+) and the depolarization of mitochondrial membrane potential were observed, dose- and time-dependently, after treatment of SCC-4 cells with various concentrations of Gyp. Gyp inhibited the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xl, but promoted the levels of the pro-apoptotic protein Bax. Western blotting showed the releases of cytochrome c and Endo G and both were also confirmed by confocal laser microscopic systems. The GADD153 moved to nuclei (nuclear translocation). In conclusion, Gyp induced ER stress and production of reactive oxygen species and Ca(2+), change the ratio of Bcl-2 and Bax, followed by the dysfunction of mitochondria, caused cytochrome c release, activation of caspase-3 before leading to apoptosis. These results provide information towards an understanding of the mechanisms by which Gyp induces cell cycle arrest and apoptosis in human tongue cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Extratos Vegetais/farmacologia , Neoplasias da Língua/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/patologia , Fase G1/efeitos dos fármacos , Gynostemma , Humanos , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína bcl-X/antagonistas & inibidoresRESUMO
Gypenosides (Gyp), components of Gynostemma pentaphyllum Makino, were found to induce suppression of human tongue squamous cell carcinoma SCC4 cell growth and induce apoptosis in response to overexpression of reactive oxygen species, calcium (Ca(+2)) and to decrease mitochondrial membrane potential in vitro. In this study, the effect of Gyp on cell migration and invasion of human tongue SCC4 cells was examined. SCC4 cells treated in vitro with Gyp migrated and invaded less than cells treated with phosphate-buffered saline (PBS) as a control. Gyp inhibited migration and invasion by down-regulating the production of RAS, NFkappaB, COX2, ERK1/2 and MMP-9 relative to PBS only. These results show that Gyp inhibits invasion and migration of human tongue SCC4 cells by down-regulating proteins associated with these processes, resulting in reduced metastasis.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias da Língua/enzimologia , Neoplasias da Língua/patologia , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Gynostemma , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Extratos Vegetais/farmacologia , Quinase Induzida por NF-kappaBRESUMO
Gynostemma pentaphyllum Makino is known in Asia for its effect on the treatment of hepatitis and cardiovascular diseases. Gypenosides (Gyp) are the major components extracted from Gynostemma pentaphyllum Makino. However, the molecular mechanism underlying the Gyp-induced cell cycle arrest and apoptotic process is unclear. In this study, the chemopreventive role of Gyp in human lung cancer (A549) cells in vitro was evaluated by studying the regulation of the cell cycle and apoptosis. Gyp induced GO/G1 arrest and apoptosis in the human lung cancer A549 cells. Investigation of the cyclin-dependent protein kinase inhibitors by Western blotting showed that p16, p21, p27 and p53 proteins were increased with the increasing time of incubation with Gyp in the A549 cells. This increase may be the major factor by which Gyp caused GO/G1 arrest in the examined cells. Flow cytometric assay and gel electrophoresis of DNA fragmentation also confirmed that Gyp induced apoptosis in the A549 cells. Our data demonstrated that Gyp-induced apoptotic cell death was accompanied by up-regulation of Bax, caspase-3 and caspase-9, but down-regulation of the Bcl-2 levels. Taken together, Gyp appears to exert its anticancer properties by inducing GO/GI-phase arrest and apoptosis via activation of caspase-3 in human lung A549 cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Ciclina E/antagonistas & inibidores , Fase G1/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Gynostemma , Humanos , Neoplasias Pulmonares/patologia , Modelos Biológicos , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismoRESUMO
AIM OF THE STUDY: Gypenosides, the saponins extract derived from Gynostemma pentaphyllum Makino, have been used for treating hepatitis and cancer in Asia. Our previous study demonstrates that gypenosides inhibit the onset and improve the recovery of liver fibrosis induced by CCl4 in rats. In this study, we used the isolated rat hepatic stellate cells (HSCs) as a model to study the cellular mechanism of gypenosides-inhibited liver fibrosis. MATERIALS AND METHODS: Rat HSCs was treated with PDGF, gypenosides or vehicle. Cell viability was assessed by trypan blue staining. Apoptosis and cell cycle were evaluated by flow cytometry. The activation or inhibition of signal molecules was detected by Western blotting. RESULTS: Our results showed that 500 microg/ml gypenosides decreased PDGF-induced rat HSCs numbers (8750+/-2629 versus 103,000+/-6683, p<0.001, 95% confidence interval) and arrested cells at the G1 phase without the presence of sub-G1 fraction. Analysis of PDGF-induced proliferative molecules including phosphorylation of Akt and p70 S6K, gypenosides inhibited the activation of this signal pathway. Furthermore, gypenosides down-regulated the protein expression of cell cycle G1-specific cyclin D1 and D3. CONCLUSIONS: Gypenosides inhibited PDGF-induced HSCs proliferation by inhibiting the signal pathway of PDGF-Akt-p70 S6K and down-regulation of cyclin D1 and D3 expression.
Assuntos
Fase G1/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Animais , Anexina A5/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Ciclina D1/biossíntese , Ciclina D1/genética , Ciclina D3 , Ciclinas/biossíntese , Ciclinas/genética , Fibrose , Gynostemma/química , Hepatócitos/patologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/farmacologia , Pós , Ratos , Ratos Sprague-Dawley , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
We have previously reported that gypenosides induce apoptosis in human hepatocarcinoma Huh-7 cells through a mitochondria-dependent caspase-9 activation cascade. In order to further explore the critical events leading to apoptosis in gypenosides-treated cells, the following effects of gypenosides on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MPT), and the subcellular distribution of Bcl-2 and Bax. We show that gypenosides-induced apoptosis was accompanied by the generation of intracellular ROS, disruption of MPT, and inactivation of ERK, as well as an increase in mitochondrial Bax and a decrease of mitochondrial Bcl-2 levels. Ectopic expression of Bcl-2 or treatment with furosemide attenuated gypenosides-triggered apoptosis. Treatment with ATA caused a drastic prevention of apoptosis and the gypenosides-mediated mitochondrial Bcl-2 decrease and Bax increase, but failed to inhibit ROS generation and MPT dysfunction. Incubation with antioxidants significantly inhibited gypenosides-mediated ROS generation, ERK inactivation, MPT and apoptosis. Moreover, an increase of the intracellular calcium ion (Ca(2+)) concentration rapidly occurred in gypenosides-treated Huh-7 cells. Buffering of the intracellular Ca(2+) increase with a Ca(2+) chelator BAMTA/AM blocked the gypenosides-elicited ERK inactivation, ROS generation, Bcl-2/Bax redistribution, mitochondrial dysfunction, and apoptosis. Based on these results, we propose that the rise in intracellular Ca(2+) concentration plays a pivotal role in the initiation of gypenosides-triggered apoptotic death.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Gynostemma , Fitoterapia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The effects of the gypenosides (Gyp), a component of Gynostemma pentaphyllum Makino, on the cell viability, cell cycle and induction of apoptosis were investigated in human colon cancer colo 205 cells. Gyp was cytotoxic to colo 205 cells with an IC50 of 113.5 microg/ml. The decreasing number of viable cells appeared to be due to the induction of cell cycle arrest and apoptotic cell death, since Gyp induced morphological changes and internucleosomal DNA fragmentation and increased the sub-G1 group. The production of reactive oxygen species and Ca2+ and the depolarization of mitochondrial membrane potential were observed, dose- and time-dependently, after treatment with various concentrations of Gyp. Gyp treatment also gradually decreased the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl, but increased the expression of the pro-apoptotic protein Bax. Gyp increased the levels of p53 and promoted the release of cytochrome c and the activation of caspase-3 before leading to apoptosis. These results provide information towards an understanding of the mechanisms by which Gyp induces cell cycle arrest and apoptosis in human colon cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Gynostemma , Humanos , Microscopia de Fluorescência , Extratos Vegetais/farmacologiaRESUMO
Ginsenoside Rh2 (Rh2), a purified ginseng saponin, has been shown to have antiproliferative effects in certain cancer cell types. However, the molecular mechanisms of Rh2 on cell growth and death have not been fully clarified. In this study, the antiproliferative effect of Rh2 in human lung adenocarcinoma A549 cells was investigated. Treatment of A549 cells with 30 mug/ml Rh2 resulted in G(1) phase arrest, followed by progression to apoptosis. This Rh2-mediated G(1) arrest was accompanied by downregulation of the protein levels and kinase activities of cyclin-D1, cyclin-E and Cdk6, and the upregulation of pRb2/p130. In addition, Rh2-induced apoptosis was confirmed by TUNEL assay and DNA fragmentation analysis. Administration of Rh2 caused an increase in the expression levels of TRAIL-RI (DR4) death receptor but did not alter the levels of other death receptors or Bcl-2 family molecules. Furthermore, the Rh2-induced apoptosis was significantly inhibited by DR4:Fc fusion protein, which inhibits TRAIL-DR4-mediated apoptosis. In addition, caspase-2, caspase-3 and caspase-8 were highly activated upon Rh2 treatment. Inhibitors of caspase-2, caspase-3 and caspase-8 markedly prevented the cell death induced by Rh2. Inhibitor of caspase-8 significantly inhibited the activation of caspase-2, caspase-3 and caspase-8. These observations indicate that multiple G(1)-related cell cycle regulatory proteins are regulated by Rh2 and contribute to Rh2-induced G(1) growth arrest. The increase in the expression level of DR4 death receptor may play a critical role in the initiation of Rh2-triggered apoptosis, and the activation of the caspase-8/caspase-3 cascade acts as the executioner of the Rh2-induced death process.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Ginsenosídeos/farmacologia , Adenocarcinoma/patologia , Caspases/metabolismo , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Fatores de TempoRESUMO
Liver fibrosis is an over-accumulation of extra-cellular matrix (ECM) and the hepatic stellate cell (Ito cell) play a central role in the pathogenesis of liver fibrosis. There are a lot of growth factors and cytokines involved in the activation of hepatic stellate cell, including of transforming growth factor (TGF-alpha, TGF-beta1), platelet-derived growth factor (PDGF), interleukin (IL-1alpha,beta, IL-6) and tumor necrosis factor (TNF-alpha). Sho-saiko-to (TJ-9; Xiao-Chai-Hu-Tang in Chinese) was the most popular herbal medicine for the treatment of chronic liver disease in Chinese and Japanese. Our aim of the current study was to examine whether TJ-9 regulated the growth factors and cytokines in the fibrogenesis of bile duct ligated model. Therefore, we assessed the TJ-9's potential in regulating TGF-beta1, PDGF mRNA expression, the amount of IL-1alpha, IL-1beta, IL-6, TNF-alpha and the fibrotic marker "PIII NP" in the serum. Then, using the immunohistochemical stain to observe the TGF-beta1 expression in the tissue. Our results showed that TJ-9 at a dose of 0.5 g/(kgday) significantly reduced the serum level of PIII NP, the mRNA expression of TGF-beta1 and PDGF. For the cytokines involved in the activation of Ito cell, TJ-9 at a dose of 0.5 g/(kgday) significantly suppressed the increasing tendency of IL-1beta and enhanced the production of TNF-alpha. Finally, we concluded that: (1) TJ-9 at a dose of 0.5g/(kgday) significantly reduced the serum fibrotic marker PIII NP in the bile duct ligated model, and its mechanism was partly by means of downregulating the mRNA of TGF-beta1 and PDGF. These results also confirmed by the immunohistochemical staining of TGF-beta1. (2) TJ-9 at a dose of 0.5 g/(kgday) suppressed the increasing tendency of IL-1beta and stimulated the production of TNF-alpha to inhibit Ito cell proliferation and collagen formation.
Assuntos
Ductos Biliares/efeitos dos fármacos , Citocinas/fisiologia , Medicamentos de Ervas Chinesas/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Fator de Crescimento Transformador beta/fisiologia , Animais , Ductos Biliares/fisiologia , Medicamentos de Ervas Chinesas/farmacologia , Cirrose Hepática/metabolismo , Masculino , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta1RESUMO
Hepatic fibrosis is an over-accumulation of extracellular matrix (ECM). It is a result of an imbalance between collagen synthesis and degradation. Matrix metalloproteinase (MMP) has degradative activity against collagen, but tissue inhibitors of metalloproteinase (TIMP) control the active forms of MMP by blocking the active site of MMP. In our study, we established the bile duct ligated model (BDL) in rats to evaluate anti-fibrotic potential of Chinese medicine sho-saiko-to (TJ-9). We assessed the drug's potential in inhibiting collagen accumulation, suppressing procollagen alpha1 types (I) and (III), and TIMP-1 mRNA expression. After administration of TJ-9, hyperbilirubinemia reduced approximately four-fold when compared with BDL-untreated group. TJ-9 also significantly reduced the collagen content and fibrogenic score, as well as downregulated elevated procollagen alpha1 types (I) and (III) and TIMP-1 mRNA level. Finally, we concluded that (1) TJ-9 significantly reduced cholestasis in rats with BDL, (2) TJ-9 markedly reduced the collagen content by 50%, and (3) TJ-9 exerted its antifibrogenic effect by downregulation of the mRNA expression of procollagen alpha1 types (I) and (III), and TIMP-1 in liver tissue.
Assuntos
Colágeno Tipo III/biossíntese , Colágeno Tipo I/biossíntese , Colágeno/farmacocinética , Medicamentos de Ervas Chinesas/farmacologia , Cirrose Hepática/prevenção & controle , Animais , Ductos Biliares/cirurgia , Modelos Animais de Doenças , Regulação para Baixo , Ligadura , Cirrose Hepática/veterinária , Masculino , RNA Mensageiro/análise , Ratos , Ratos Wistar , Inibidor Tecidual de Metaloproteinase-1/biossínteseRESUMO
N-acetylation plays an important role in the metabolism of arylamine drugs and carcinogens and is catalyzed by cytosolic N-acetyltransferase (NAT). Gypenosides are the major components of Gynostemma pentaphyllum Makino which had been used as a natural folk medicine in the Chinese populations. Gypenosides were selected for examining the inhibition on the N-acetylation of 2-aminofluorene (AF), DNA-AF adduct formation and NAT gene expression in the human cervix epithelioid carcinoma cell line (HeLa). Various concentrations of gypenosides were individually added to the culture medium of human cervix epithelioid carcinoma cells (HeLa). The N-acetylation of AF was determined by high performance liquid chromatography (HPLC) assaying for the amounts of acetylated 2-aminofluorene (AAF) and nonacetylated 2-aminofluorene (AF). The N-acetylation of AF in the human HeLa cancer cells was suppressed by gypenosides in a dose-dependent manner. The data also demonstrated that gene expression (NAT1 mRNA) of NAT in human cervix epithelioid carcinoma cells (HeLa) was inhibited and decreased by gypenosides. After the incubation of HeLa cells with 30 or 60 microM AF and with or without 350 microg/ml gypenosides cotreatment, DNA was isolated and hydrolyzed to nucleotides, adducted nucleotides were extracted into butanol and analyzed DNA-AF adducts by HPLC. The data demonstrated that gypenosides decrease the levels of DNA-AF adduct formation in HeLa cells.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Adutos de DNA/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/toxicidade , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Carcinógenos/análise , Carcinógenos/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura/farmacologia , Adutos de DNA/biossíntese , Relação Dose-Resposta a Droga , Fluorenos/análise , Fluorenos/metabolismo , Gynostemma/toxicidade , Células HeLa , Humanos , RNA Mensageiro/metabolismoRESUMO
Shao-Fu-Zhu-Yu-Tang (SFZYT) is reportedly beneficial to sperm. In this study, we examined sperm acrosomal activity and serum free radical changes to evaluate the possible mechanism of SFZYT. A clinical study evaluated the sperm count and motility in 36 patients with chronic prostatitis before and after treatment for 60 days. The results revealed a significant increase in sperm motility after treatment as evaluated by computer-assisted semen analysis (17.27 +/- 9.00 versus 28.29 +/- 10.00, p < 0.01). An increase in sperm quantity and quality was observed by count and morphology with a high-powered intravital microscope. To gain an understanding of the mechanisms that caused this effect, we assessed sperm acrosin activity levels before (10.6 micro lu/10(6)) and after medication (28.6 micro lu/10(6)) (p < 0.01). The levels of the free radicals was relatively higher before medication, 2144, compared to a normal value of 780 after medication (p < 0.01). In conclusion, SFZYT increased the motility and quality of human semen and this increase is related to an increase in sperm acrosin activity. SFZYT also works as a sperm antioxidant and antiaging agent.
Assuntos
Antioxidantes/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Fitoterapia , Plantas Medicinais , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Acrosina/metabolismo , Adulto , Antioxidantes/administração & dosagem , Antioxidantes/uso terapêutico , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Masculino , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Espermatozoides/metabolismoRESUMO
Norcantharidin (NCTD) is an anticancer drug routinely used against hepatoma in China. Previously, we reported that NCTD could induce mitotic arrest and apoptosis in human hepatoma HepG2 cells. However, the intracellular signaling pathways involved in NCTD-induced apoptotic cell death are still obscure. Caspase inhibitors were used to clarify the role of specific caspase in NCTD-triggered apoptotic process. Results showed that activation of caspase-9/caspase-3 cascade is required for NCTD-induced apoptotic death. To decipher the upstream signals for NCTD-induced apoptosis, we characterized the involvement of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38MAPK. The role of their downstream targets, transcription factors activating protein-1 (AP-1), and nuclear factor kappaB (NF-kappaB) in NCTD-induced apoptosis was also analyzed. Immunoblot analyses and in vitro kinase assay demonstrated that NCTD-induced apoptosis was accompanied by the elevations of the levels of phosphorylated form and kinase activity of ERK and JNK, but not p38MAPK. The inhibitor of ERK pathway (U0126 or PD98059) or JNK pathway (SP600125) markedly prevented kinase activation, and also greatly reduced NCTD-induced apoptotic cell death. Increased DNA-binding activity of AP-1 and NF-kappaB was also observed after NCTD treatment. Inhibition of NF-kappaB activation by PDTC or inhibition of AP-1 activation by curcumin drastically blocked NCTD-induced cell death. These results imply that activation of ERK and JNK, and modulation of downstream transcription factors NF-kappaB and AP-1, may be involved in NCTD-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Apoptose/fisiologia , Carcinoma Hepatocelular/enzimologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Neoplasias Hepáticas/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The effects of curcumin on the N-acetyltransferase (NAT) activity, AF-DNA adduct formation and NAT gene expression were examined using the human colon tumor cell line (colo 205). Various concentrations of curcumin were added to the cytosols or to the medium of human colon tumor cells. The NAT activity was determined by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene (AAF) and p-aminobenzoic acid (N-Ac-PABA) and nonacetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). The NAT activity in the human colon tumor cells and cytosols was suppressed by curcumin in a dose-dependent manner. The results demonstrated that gene expression (NAT1 mRNA) in human colon tumor cells was inhibited by curcumin. The apparent values of Km and Vmax of NAT of human colon tumor cells were also decreased by curcumin in cytosols. Curcumin may act as a noncompetitive inhibitor. After the incubation of human colon tumor cells with AF with or without curcumin cotreatment, the cells were recovered and DNA was prepared, hydrolyzed to nucleotides, the adducted nucleotides were extracted into butanol and AF-DNA adducts analyzed by HPLC. The results also demonstrated that when curcumin was added to the media a decrease in AF-DNA adduct formation was seen in the human colon tumor cells. The finding of AF-DNA adduct formation in cultured human colon tumor cells suggests the usefulness of cultured cells for assessing arylamine-induced DNA damage.