RESUMO
BACKGROUND/AIM: Lung cancer notably contributes to tumor-associated mortality worldwide, and standard chemotherapy is used for lung cancer patients. However, its therapeutic efficacy remains unsatisfactory. This study aimed to evaluate the effects and molecular mechanisms of sorafenib and bufalin combination therapy on lung cancer cells in vitro. MATERIALS AND METHODS: NCI-H292 cells were treated with sorafenib, bufalin, and sorafenib in combination with bufalin. Cell viability, ROS production, Ca2+ release, and mitochondrial membrane potential were examined by flow cytometric assay. Annexin V/PI staining and chromatin condensation were examined by the apoptosis assays. Finally the molecular mechanism of apoptosis-associated protein expression was investigated by western blotting. RESULTS: NCI-H292 cells treated with sorafenib in combination with bufalin showed significantly decreased viability, enhanced cellular apoptosis, and DNA condensation when compared to that with sorafenib or bufalin alone. Moreover, the combination treatment exhibited higher reactive oxygen species (ROS) production and lower mitochondrial membrane potential (ΔΨm). The combined treatment resulted in higher expression of SOD but lower catalase compared to sorafenib treatment alone. Compared to sorafenib or bufalin treatment alone, the combination treatment resulted in lower Bcl-2 expression but higher Bax, Bad, APAF-1, caspase-3, and caspase-9. CONCLUSION: Sorafenib in combination with bufalin shows more potent cytotoxic effects and cell apoptosis than sorafenib or bufalin treatment alone in NCI-H292 cells. The combined treatment significantly enhanced apoptotic cell death in NCI-H292 lung cancer cells by activating ROS-, mitochondria-, and caspase-signaling pathways in vitro.
Assuntos
Apoptose , Neoplasias Pulmonares , Bufanolídeos , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Sorafenibe/farmacologiaRESUMO
Demethoxycurcumin (DMC), a derivate of curcumin, has been shown to induce apoptotic cell death in human glioblastoma multiforme GBM 8401 cells via cell cycle arrest and induction of cell apoptosis. However, there is no report showing DMC suppresses glioblastoma multiforme cells in vivo. In the present study, we investigated the effects of DMC on GBM8401 cells in vivo. At first, we established a luciferase-expressing stable clone named GBM 8401/luc2. Second, mice were inoculated subcutaneously with GBM 8401/luc2 cells to generate a xenograft tumor mice model. After inoculation, tumor volume reached 100-120 mm3, and all mice were randomly divided into three groups: Group I was treated with 110 µL phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 30 mg/kg of DMC, and Group III with 60 mg/kg of DMC. Mice from each group were given the oral treatment of DMC by gavage for 21 days. The body weight and tumor volume were recorded every 3 days. DMC significantly decreased the tumor volumes, and 60 mg/kg treatment showed a higher decrease in tumor volumes than that of 30 mg/kg, However, DMC did not affect the body weights. The photons emitted from mice tumors were detected with Xenogen IVIS imaging system, DMC at both doses decreased the total photon flux and 60 mg/kg treatment of DMC has low total photon flux than that of 30 mg/kg. The tumor volumes and weights in 60 mg/kg treatment of DMC were lower than that of 30 mg/kg. Immunohistochemical analysis was used to measure protein expression of tumors and results showed that DMC treatment led to lightly staining with anti-Bcl-2 and -XIAP and 60 mg/kg treatment of DMC has lighter staining with anti-Bcl-2 and -XIAP than that of 30 mg/kg. The higher dose (60 mg/kg) of DMC has higher signals of cleaved-caspase-3 than that of the lower dose (30 mg/kg). Furthermore, the hematoxylin and eosin (H&E) staining of liver tissues showed no significant difference between DMC-treated and control-groups. Overall, these observations showed that DMC suppressed tumor properties in vivo and DMC may be used against human glioblastoma multiforme in the future.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Diarileptanoides/uso terapêutico , Glioblastoma/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Diarileptanoides/toxicidade , Genes Reporter , Glioblastoma/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Distribuição Aleatória , Carga Tumoral , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/análise , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/análiseRESUMO
Fisetin, a naturally occurring flavonoid, is found in common fruits and vegetables and has been shown to induce cytotoxic effects in many human cancer cell lines. No information has shown that fisetin induced cell cycle arrest and apoptosis in mouse leukemia WEHI-3 cells. We found that fisetin decreased total viable cells through G0/G1 phase arrest and induced sub-G1 phase (apoptosis). We have confirmed fisetin induced cell apoptosis by the formation of DNA fragmentation and induction of apoptotic cell death. Results indicated that fisetin induced intracellular Ca 2+ increase but decreased the ROS production and the levels of ΔΨ m in WEHI-3 cells. Fisetin increased the activities of caspase-3, -8 and -9. Cells were pre-treated with inhibitors of caspase-3, -8 and -9 and then treated with fisetin and results showed increased viable cell number when compared to fisetin treated only. Fisetin reduced expressions of cdc25a but increased p-p53, Chk1, p21 and p27 that may lead to G0/G1 phase arrest. Fisetin inhibited anti-apoptotic protein Bcl-2 and Bcl-xL and increased pro-apoptotic protein Bax and Bak. Furthermore, fisetin increased the protein expression of cytochrome c and AIF. Fisetin decreased cell number through G0/G1 phase arrest via the inhibition of cdc25c and induction of apoptosis through caspase-dependent and mitochondria-dependent pathways. Therefore, fisetin may be useful as a potential therapeutic agent for leukemia.
Assuntos
Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Flavonoides/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Leucemia/genética , Leucemia/patologia , Animais , Flavonóis , Camundongos , Células Tumorais CultivadasRESUMO
The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G2/M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca 2+ production and decreased the levels of ΔΨm . Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1 α , IRE-1 ß , caspase-4, ATF-6 α , Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Bufanolídeos/farmacologia , Mitocôndrias/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Extratos de Tecidos/farmacologia , Bufanolídeos/química , Bufanolídeos/isolamento & purificação , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Carcinoma Nasofaríngeo/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/metabolismo , Extratos de Tecidos/isolamento & purificação , Células Tumorais CultivadasRESUMO
Gypenosides (Gyp), the primary components of Gynostemma pentaphyllum Makino, have long been used as a Chinese herbal medicine. In the present study, the effects of Gyp on cell viability, the cell cycle, cell apoptosis, DNA damage and chromatin condensation were investigated in vitro using human oral cancer HSC-3 cells. The results of the present study indicated that Gyp induces cell death, G2/M phase arrest and apoptosis in HSC-3 cells in a dose-dependent manner. It was also demonstrated that Gyp decreased the depolarization of mitochondrial membrane potential in a time-dependent manner. A cDNA microarray assay was performed and the results indicated that a number of genes were upregulated following Gyp treatment. The greatest increase was a 75.42-fold increase in the expression of GTP binding protein in skeletal muscle. Levels of the following proteins were also increased by Gyp: Serpine peptidase inhibitor, clade E, member 1 by 20.25-fold; ras homolog family member B by 18.04-fold, kelch repeat and BTB domain containing 8 by 15.22-fold; interleukin 11 by 14.96-fold; activating transcription factor 3 by 14.49-fold; cytochrome P450, family 1 by 14.44-fold; ADP-ribosylation factor-like 14 by 13.88-fold; transfer RNA selenocysteine 2 by 13.23-fold; and syntaxin 11 by 13.08-fold. However, the following genes were downregulated by GYP: Six-transmembrane epithelial antigen of prostate family member 4, 14.19-fold; γ-aminobutyric acid A receptor by 14.58-fold; transcriptional-regulating factor 1 by 14.69-fold; serpin peptidase inhibitor, clade B, member 13 by 14.71-fold; apolipoprotein L 1 by 14.85-fold; follistatin by 15.22-fold; uncharacterized LOC100506718; fibronectin leucine rich transmembrane protein 2 by 15.61-fold; microRNA 205 by 16.38-fold; neuregulin 1 by 19.69-fold; and G protein-coupled receptor 110 by 22.05-fold. These changes in gene expression illustrate the effects of Gyp at the genetic level and identify potential targets for oral cancer therapy.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Bufalin, a component of Chan Su (frog), has been shown to have biological activities including anti-tumor effects. Gefitinib has been used as an anti-cancer drug in lung cancer patients; however, some patients eventually become gefitinib resistant. AIM OF THE STUDY: In this study, we investigated anti-metastasis effects of bufalin in gefitinib resistant NCI-H460 lung cancer cells. MATERIALS AND METHODS: The effects of the bufalin in gefitinib resistant NCI-H460 lung cancer cells were investigated on cell viability using flow cytometry. The adhesion capacity, wound healing assay, invasion and migration assay, and Western blot analysis were used to understand the molecular mechanisms in this study RESULTS: Under sub-lethal concentrations (from 2.5 up to 10nM), bufalin significantly inhibits cell adhension, migration and invasion nature of gefitinib resistant H460 cells. Western blotting assay revealed that bufalin depressed some of the key metastasis-related proteins, such as SOS-1, MMP-2 and Rho A underwent significant reduction. Phosphorylated Focal adhesion kinase (p-FAK), phosphorylated extracellular signal-regulated kinase (p-ERK1/2), Ras and E-cadherin were significantly reduced at 48h treatment. However, phosphorylated p38 (p-p38), phosphorylated c-Jun NH2-terminal kinase (p-JNK1/2) and NF-κBp65 were increased. CONCLUSIONS: Based on these observations, we suggest that bufalin can be used in anti-metastasis of gefitinib resistant NCI-H460 lung cancer cells in the future.
Assuntos
Bufanolídeos/farmacologia , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Invasividade Neoplásica/patologia , Quinazolinas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gefitinibe , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteína SOS1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.
Assuntos
Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Fase G2/efeitos dos fármacos , Isotiocianatos/química , Fitoterapia , Extratos Vegetais/farmacologia , Anexina A5/genética , Anexina A5/metabolismo , Apoptose/genética , Cálcio/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular/genética , Divisão Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Estresse do Retículo Endoplasmático/genética , Fase G2/genética , Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Estimulação Química , SulfóxidosRESUMO
Polygonum cuspidatum is a traditional Chinese herbal medicine used in the treatment of various diseases. In the present study, we investigated whether the crude extract of Polygonum cuspidatum (CEPC) could affect immune responses of murine leukemia cells in vivo. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemic mice and then were treated orally with CEPC at 0, 50, 100 and 200 mg/kg for three weeks. Animals were weighed and blood, liver, spleen samples were collected for further analyses. Results indicated that CEPC did not significantly affect the body and liver weight of animals, but reduced the weight of spleen when compared to control groups. Flow cytometric assay demonstrated that CEPC increased the percentage of CD3- (T-cell marker) and CD19- (B-cell marker) positive cells, but reduced that of CD11b-positive ones (monocytes). However, it did not significantly affect the proportion of Mac-3-positive cells (macrophages), compared to control groups. Results indicated that CEPC promoted phagocytosis by macrophages from blood samples at all examined doses but did not affect that of macrophages from the peritoneal cavity. CEPC also promoted natural killer cell activity of splenocytes at 200 mg/kg of CEPC. CEPC promoted B-cell proliferation at 200 mg/kg treatment when cells were stimulated with lipopolysaccharides but did not promote T-cell proliferation at three doses of CEPC treatment on concanavalin A stimulation.
Assuntos
Fallopia japonica/química , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Extratos Vegetais/farmacologia , Animais , Antígenos de Superfície/metabolismo , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacologia , Leucemia Experimental , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Extratos Vegetais/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Crude extract of Rheum palmatum L. (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the anti-metastasis effects of CERP on LS1034 human colorectal cancer cells in vitro and examined potential mechanisms of its effects. CERP significantly inhibited cell migration and invasion of LS1034 cells. We also found that CERP inhibited protein levels of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9), and cytosolic NF-kB p65, RHO A, ROCK 1. Furthermore, we found CERP inhibited protein levels of GRB2, SOS1, MKK7, FAK, Rho A, ROCK 1, VEGF, PKC, AKT, phosphor-AKT (Thr308), Cyclin D, iNOS, COX2, NF-kB p65, p-ERK1/2, p-JNK1/2, p-p38, p-c-jun, MMP-2, MMP-9, MMP-1, MMP-7, MMP-10, UPA and increased the protein level of Ras in LS1034 cells. In conclusion, our results suggest that CERP may be used as a novel anti-metastasis agent for the treatment of human colon cancer cells.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Extratos Vegetais/farmacologia , Rheum/química , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Extratos Vegetais/química , Rheum/metabolismo , Fator de Transcrição RelA/metabolismo , Cicatrização/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Crude extract of Rheum palmatum L (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the effects of CERP on LS1034 human colorectal cancer cells in vitro and also examined possible mechanisms of cell death. Flow cytometric assays were used to measure the percentage of viable cells, cell cycle distribution including the sub-G1 phase (apoptosis), the activities of caspase-8, -9, and -3, reactive oxygen species (ROS) and Ca(2+) levels, and mitochondrial membrane potential (ΔΨm). DNA damage, nuclei condensation, protein expression, and translocation were examined by Comet assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, Western blotting, and confocal laser system microscope, respectively. CERP induced apoptosis as seen by DNA fragmentation and DAPI staining in a concentration- and time-dependent manner in cancer cells. CERP was associated with an increase in the Bax/Bcl-2 protein ratio and CERP promoted the activities of caspase-8, -9, and -3. Both ROS and Ca(2+) levels were increased by CERP but the compound decreased levels of ΔΨm in LS1034 cells. Laser confocal microscope also confirmed that CERP promoted the expressions of AIF, Endo G, cytochrome c, and GADD153 to induce apoptosis through mitochondrial-dependent pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias do Colo/patologia , Extratos Vegetais/farmacologia , Rheum/química , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Ensaio Cometa , Citocromos c/metabolismo , Dano ao DNA , Humanos , Indóis/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Chlorogenic acid exists widely in edible and medicinal plants and acts as an antioxidant. It is known to exert antitumor activity via induction of apoptosis in many human cancer cells. However, its signaling pathway in human leukemia cells still remains unclear. Therefore, we investigated the roles of reactive oxygen species (ROS), mitochondria and caspases during chlorogenic acid-induced apoptosis of U937 human leukemia cells. Chlorogenic acid exhibited a strong cytotoxicity and induced apoptosis in U937 cells, as determined by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Chlorogenic acid induced apoptosis by promoting ROS production and reduced the mitochondrial membrane potential (ΔΨm), as assayed by flow cytometry. Furthermore, the activity of caspase-3 was evaluated and results indicated that chlorogenic acid promoted caspase-3 activity in U937 cells. Results from western blot analysis showed that chlorogenic acid promoted expression of caspase-3, -7, -8 and -9 in U937 cells. Taken together, these results suggest that chlorogenic acid may induce apoptosis by reducing the levels of ΔΨm and by increasing the activation of caspase-3 pathways in human leukemia U937 cells in vitro.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Clorogênico/farmacologia , Leucemia , Mitocôndrias , Caspase 3/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células U937RESUMO
Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca(2+) productions as well as loss of mitochondrial membrane potential (ΔΨ(m)) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca(2+) release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/patologia , Emodina/farmacologia , Adenocarcinoma/metabolismo , Animais , Sequência de Bases , Cálcio/metabolismo , Caspase 3/genética , Caspase 9/genética , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Primers do DNA , Humanos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
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
The natural antioxidant gallic acid (GA) has demonstrated a significant inhibition of cell proliferation and induction of apoptosis in a series of cancer cell lines. However, there is no available information to show whether GA induces apoptosis in human skin cancer cells. In the present study, we report GA-induced apoptosis in A375.S2 human melanoma cells. GA affected morphological changes, decreased the percentage of viable cells and induced apoptosis in A375.S2 cells in a dose- and time-dependent manner. Observation of the molecular mechanism of apoptosis in A375.S2 cells showed that GA up-regulated the proapoptotic proteins such as Bax, and induced caspase cascade activity, but down-regulated antiapoptotic proteins such as Bcl-2. GA induced reactive oxygen species (ROS) and intracellular Ca2+ productions and decreased the level of mitochondrial membrane potential (DeltaPsim) in A375.S2 cells in a time-dependent manner. GA triggered cytosolic release of apoptotic molecules, cytochrome c, promoted activation of caspase-9 and caspase-3, and ultimately apoptotic cell death. In addition, GA also promoted cytosolic release of apoptosis-inducing factor (AIF) and endonuclease G (Endo G). Therefore, GA may also induce apoptosis through a caspase-independent pathway. Our results suggest that GA might be a potential anticancer compound; however, in depth in vivo studies are needed to elucidate the exact mechanism.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/fisiologia , Ácido Gálico/farmacologia , Melanoma/patologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Melanoma/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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
Emodin, aloe-emodin and rhein are major compounds in rhubarb (Rheum palmatum L.), used in Chinese herbal medicine, and found to have antitumor properties including cell cycle arrest and apoptosis in many human cancer cells. Our previous studies also showed that emodin, aloe-emodin and rhein induced apoptosis in human tongue cancer SCC-4 cells. However, the detail regarding emodin, aloe-emodin and rhein affecting migration and invasion in SCC-4 cells are not clear. In the present study, we investigated whether or not emodin, aloe-emodin and rhein inhibited migration and invasion of SCC-4 cells. Herein, we demonstrate that emodin, aloe-emodin and rhein inhibit the protein levels and activities of matrix metalloproteinase-2 (MMP-2) but did not affect gene expression of MMP-2, however, they inhibited the gene expression of MMP-9 and all also inhibited the migration and invasion of human tongue cancer SCC-4 cells. MMP-9 (gelatinase-B) plays an important role and is the most associated with tumor migration, invasion and metastasis in various human cancers. Results from zymography and Western blotting showed that emodin, aloe-emodin and rhein treatment decrease the levels of MMP-2, urokinase plasminogen activator (u-PA) in a concentration-dependent manner. The order of inhibition of associated protein levels and gene expression of migration and invasion in SCC-4 cells are emodin >aloe-emodin >rhein. Our results provide new insight into the mechanisms by which emodin, aloe-emodin and rhein inhibit tongue cancers. In conclusion, these findings suggest that molecular targeting of MMP-9 mRNA expression by emodin, aloe-emodin and rhein might be a useful strategy for chemo-prevention and/or chemo-therapeutics of tongue cancers.
Assuntos
Aloe/química , Antraquinonas/farmacologia , Emodina/farmacologia , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/biossíntese , Neoplasias da Língua/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Invasividade Neoplásica , RNA Mensageiro/metabolismo , Neoplasias da Língua/tratamento farmacológico , Ativador de Plasminogênio Tipo Uroquinase/biossínteseRESUMO
Agaricus blazei Murill (ABM) has shown particularly strong results in treating and preventing cancer and has also traditionally been used as a food source in Brazil. However, the exact immune responses regarding the phagocytosis of macrophage and, the activity of natural killer (NK) cells in normal mice after exposure to ABM extract was unclear. The goal of this study was to investigate whether or not ABM extract can promote immune responses in normal BALB/c mice. BALB/c mice were treated with different doses of ABM extract for different time periods. The results indicated that ABM extract significantly promoted the proliferation of splenocytes both in vitro and in vivo. ABM extract promoted the levels of interleukein-6 (IL-6) and, interferon-gamma (IFN-gamma) but reduced the levels of IL-4 in vitro and in vivo. The percentage of macrophages with phagocytosis after ABM extract treatment increased and these effects were of dose-dependent manners, both in vitro and in vivo. YAC-1 target cells were killed by NK cells from the mice after treatment with ABM extract at 3 and 6 mg/kg/day for up to 14 days at target cell ratios of 25:1 and 50:1. Taken together, these results show that ABM extract promoted immunomodulations in normal BALB/c mice in vitro and in vivo.