RESUMO
Melanin produced by melanocytes protects our skin against ultraviolet (UV) radiation-induced cell damage and oxidative stress. Melanin overproduction by hyperactivated melanocytes is the direct cause of skin hyperpigmentary disorders, such as freckles and melasma. Exploring natural whitening agents without the concern of toxicity has been highly desired. In this study, we focused on a Bifidobacterium longum strain, ZJ1, isolated from a Chinese centenarian, and we evaluated the anti-melanogenic activity of the distinctive extracts of ZJ1. Our results demonstrated that whole lysate (WL) and bacterial lysate (BL) of ZJ1 ferments efficiently reduce α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16-F10 cells as well as the melanin content in zebrafish embryos. BL and WL downregulate melanogenesis-related gene expression and indirectly inhibit intracellular tyrosinase activity. Furthermore, they both showed antioxidant activity in a menadione-induced zebrafish embryo model. Our results suggest that ZJ1 fermentation lysates have application potential as therapeutic reagents for hyperpigmentary disorders and whitening agents for cosmetics.
Assuntos
Antioxidantes , Bifidobacterium longum , Clareadores , Hiperpigmentação , Melaninas , Animais , Humanos , Antioxidantes/farmacologia , Bifidobacterium longum/isolamento & purificação , Bifidobacterium longum/metabolismo , Centenários , População do Leste Asiático , Hiperpigmentação/tratamento farmacológico , Hiperpigmentação/metabolismo , Melaninas/metabolismo , Peixe-Zebra , Idoso de 80 Anos ou maisRESUMO
Lung cancer has high mortality and incidence rates in which non-small cell lung cancer (NSCLC) is the primary type of lung cancer that accounts for about 80%-85% of total patients. It has been demonstrated that microRNAs (miRNAs) are critical in the incidence and progression of tumors, while the role and inner mechanism of miR-200a-3p, one type of essential miRNAs, in NSCLC have yet to be revealed. Herein, we investigated the in vitro and vivo pro-/antiproliferative influence of miR-200a-3p on NSCLC cells and utilized bioinformatic programs to further predict the SOX17 gene as miR-200a-3p's potential target. A double luciferase reporter gene experiment was performed to confirm that miR-200a-3p interacts with the SOX17 3'-UTR region specifically. On the basis of the results of Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), miR-200a-3p impacted the posttranscriptional levels of SOX17 rather than influencing its mRNA expression. In the end, we found that overexpressed SOX17 can reverse miR-200a-3p's impact on NSCLC cell proliferation and metastasis. Therefore, this study demonstrated that miR-200a-3p influences NSCLC cell proliferation and metastasis by modulating the levels of SOX17.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismoRESUMO
High-mobility group box 1 (HMGB1), a highly conserved chromosome protein, is considered as a potential therapeutic target and novel biomarker because of its regulation in the proliferation and metastasis of Hepatocellular carcinoma (HCC). Calenduloside E (CE), a natural active product, has been reported to anti-cancer effect. However, the role and underlying molecular mechanism of CE in HCC is still unclear. The purpose of this study is to investigate the effects of CE on the proliferation and migration of HCC, and then explore the possible underlying molecular mechanism. HepG2 cells were treated with CE or transfected with HMGB1 shRNA plasmids, EdU and colony formation assays were used to detect cell proliferation ability. Wound healing and transwell assays were used to determine the role of CE in cell migration. The expression of Cyclins, PCNA, MMPs, HMGB1, N-cadherin, E-cadherin and phosphorylation of p38, ERK and JNK were all detected using Western blotting. Our results showed that CE inhibited HepG2 cells proliferation and migration in a dose dependent manner; reduced the expression levels of Cycins, PCNA, HMGB1, MMPs and N-cadherin; up-regulated E-cadherin expression; enhanced the phosphorylation of p38 and JNK signalling pathways. Blocking the activation of p38 and JNK obviously reversed CE-mediated inhibitory effects on HepG2 cell proliferation and migration; reversed CE-induced down-regulation of Cyclins, PCNA, MMPs, N-cadherin and HMGB1, as well as E-cadherin up-regulation. In conclusion, our study suggested that CE reduces the expression levels of Cyclins, MMPs and epithelial-mesenchymal transformation (EMT) through p38/JNK-HMGB1 signaling axis and then inhibits HepG2 cells proliferation and migration in HepG2 cells. This study provides a new perspective for the anti-tumour molecular mechanism of CE in HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Proteína HMGB1/metabolismo , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma Hepatocelular/tratamento farmacológico , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Ácido Oleanólico/farmacologia , FitoterapiaRESUMO
Lung cancer (LC) is one of the leading causes of cancer-related death in the world. miR-24-3p plays critical roles in many cancer types, including LC. In this study, we first investigated whether miR-24-3p promoted LC cell migration and proliferation in vitro. We used three bioinformatics algorithms to predict the miR-24-3p target gene to study the molecular mechanism by which miR-24-3p contributes to LC progression. Then, we used the luciferase reporter assay to identify whether SOX7 was a direct target of miR-24-3p. Moreover, Western blotting and a quantitative real time-polymerase chain reaction analysis showed that miR-24-3p downregulated SOX7 protein expression by a post-transcriptional mechanism. Finally, we determined that SOX7 had opposing effects to those of miR-24-3p on LC cell proliferation and migration, suggesting that miR-24-3p promotes cell proliferation and migration by directly targeting SOX7. Furthermore, miR-24-3p accelerated tumor growth in xenograft mice by targeting SOX7. These results provide the first clue that miR-24-3p could play a role as an oncomiR in LC by regulating SOX7.
Assuntos
Movimento Celular , Proliferação de Células , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/metabolismo , Fatores de Transcrição SOXF/metabolismo , Células A549 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Fatores de Transcrição SOXF/genéticaRESUMO
OBJECTIVE: To investigate the effect of Nogo-66 receptor (NgR) silencing with specific small interfering RNA (siRNA) on brain injury repair in preterm rats with brain injury caused by intrauterine infection and related mechanism of action. METHODS: The pregnant Sprague-Dawley rats (with a gestational age of 15 days) were selected, and premature delivery was induced by RU486 or lipopolysaccharide (LPS). The preterm rats delivered by those treated with RU486 were selected as the control group. The preterm rats with brain injury caused by intrauterine infection induced by LPS were divided into model, empty vector, and NgR-siRNA groups, with 36 rats in each group. The rats in the control and model groups were given routine feeding only, and those in the empty vector and NgR-siRNA groups were given an injection of lentiviral empty vector or NgR-siRNA lentivirus via the lateral ventricle on postnatal day 1 (P1) and then fed routinely. On P3, P7, and P14, 8 rats in each group were randomly selected and sacrificed to harvest the brain tissue. RT-PCR was used to measure the mRNA expression of NgR. Western blot was used to to measure the protein expression of active RhoA. The immunofluorescence histochemistry was used to determine the degree of activation of microglial cells and the morphology of oligodendrocyte precursor cells (OPCs). Hematoxylin and eosin staining was used to observe the pathological changes in brain tissue. The behavioral score was evaluated on P30. RESULTS: On P3, the NgR-siRNA group had significantly lower mRNA expression of NgR and protein expression of active RhoA in brain tissue than the model and empty vector groups (P<0.05). In each group, the mRNA expression of NgR was positively correlated with the protein expression of active RhoA (P<0.05). The results of immunofluorescence histochemistry showed that on P3, the NgR-siRNA group had a significantly reduced fluorescence intensity of the microglial cells labeled with CD11b compared with the model and empty vector groups (P<0.05). The OPCs labeled with O4 antibody in the four groups were mainly presented with tripolar cell morphology. The results of pathological examination showed a normal structure of white matter with clear staining in the periventriclar area in the control group, a loose structure of white matter with disorganized fibers and softening lesions in the model and empty vector groups, and a loose structure of white matter with slightly disorganized fibers, slight gliocyte proliferation, and no significant necrotic lesions in the NgR-siRNA group. As for the behavioral score, compared with the model and empty vector groups, the NgR-siRNA group had a higher score in the suspension test, a longer total activity distance, and greater mean velocity and number of squares crossed, as well as a shorter time of slope test and a shorter time and distance of activity in the central area (P<0.05), while there were no significant differences in these parameters between the NgR-siRNA and control groups (P>0.05). CONCLUSIONS: NgR silencing with specific siRNA can effectively silence the expression of NgR in pertem rats with brain injury caused by interauterine infection and has a significant neuroprotective effect in brain injury repair.
Assuntos
Lesões Encefálicas/terapia , Receptor Nogo 1/genética , RNA Interferente Pequeno/genética , Animais , Animais Recém-Nascidos , Feminino , Inativação Gênica , Infecções/complicações , Lentivirus/genética , Masculino , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
To study the effect of sodium aescinate in inducing human breast cancer MCF-7 cells apoptosis and its possible mechanism. MTT assay was used to detect the inhibitory effect of sodium aescinate on the proliferation of MCF-7 cells. The morphological changes were observed under inverted microscope. DAPI nuclear staining was used to detect the changes in cell nucleus. Annexin V-FITC/PI flow cytometry was adopted to test the apoptosis rate. Changes in apoptosis-related proteins (PARP, cleaved caspase-8 and pro-caspase-3), cell survival-associated signal molecules (AKT and ERK) and their common upstream kinase SRC was detected by Western blotting. The result showed that after different concentrations of sodium aescinate were used to treat breast cancer MCF-7 cells, they inhibited the proliferation of MCF-7 cells in a dose-dependent manner, induced cell apoptosis (typical morphological changes in nucleus, significant increase in cell apoptosis rate). The expressions of cleaved PARP and caspase-8 increased, while the expression of pro-caspase-3 decreased, which further verified sodium aescinate's effect in inducing cell apoptosis. Sodium aescinate significantly inhibited the phosphorylation of cell survival-related signal molecules (AKT, ERK) and down-regulate the activation of their common up-stream kinase SRC. The findings indicated that sodium aescinate can block signals transiting to downstream molecules AKT, ERK, inhibit the proliferation of breast cancer cell MCF-7 cell apoptosis and induced cell apoptosis by suppressing the activation of SRC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/fisiopatologia , Medicamentos de Ervas Chinesas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Quinases da Família src/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/genéticaRESUMO
OBJECTIVE: To investigate the precise molecular mechanisms by which baicalein exerts beneficial biochemical activities in RAW264.7 macrophages treated with LPS. MATERIALS AND METHODS: RAW264.7 cells were cultured in the absence or presence of baicalein together with or without LPS. iNOS and COX-2 expression were measured by western blot and RT-PCR analyses. TNF-α, IL-1ß, and IL-6 were determined by using double-antibody sandwich ELISA. Phosphorylations of JAK1 and JAK2, and of STAT1 and STAT3 were detected by western blotting. Nuclear translocation of STAT1 and STAT3 was visualized by confocal microscopy. ROS production was detected by ROS assay. RESULTS: Baicalein significantly reduced the phosphorylation of STAT1 and STAT3 and the phosphorylation of JAK1 and JAK2, but without affecting MAPKs phosphorylation in LPS-stimulated RAW264.7 cells. Baicalein suppressed the nuclear translocation of STAT1 and STAT3 and inhibited production of iNOS upon LPS-stimulation, resulting in the inhibition of releases of NO and pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α, in a dose-dependent manner. In addition, we found that baicalein reduced the LPS-induced accumulation of ROS, confirming that baicalein serves as an antioxidant. CONCLUSIONS: Our results suggested that suppressing JAK/STATs activation and interfering with ROS production might contribute to the anti-inflammatory action of baicalein in macrophages.
Assuntos
Flavanonas/farmacologia , Inflamação/prevenção & controle , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Flavanonas/uso terapêutico , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Modelos Animais , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Oxidative stress plays an important role in neurodegenerative disorders. Ampelopsin, a flavonoid abundant in Rattan tea (Ampelopsis grossedentata), is a potent antioxidant and its neuroprotective effect against H2O2-induced apoptosis in PC12 cells is investigated here for the first time. It was found that treatment of cells with ampelopsin for 1 h significantly reduced the loss of vitality, LDH release and apoptosis and inhibited the formation of reactive oxygen species (ROS). Ampelopsin was able to prevent the activation of p38 induced by H2O2. In addition, up-regulation of heme oxygenase-1 (HO-1) expression by ampelopsin was shown to be both dose- and time-dependent. Mechanically, HO-1 expression induced by ampelopsin was found to be due to activation of the ERK and Akt signaling pathways, because it was almost completely blocked by the specific inhibitors of ERK and Akt. These results suggest that ampelopsin increases cellular antioxidant defense through activation of the ERK and Akt signaling pathways, which induces HO-1 expression and thereby protects PC12 cells from H2O2-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Heme Oxigenase (Desciclizante)/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the inhibitory effects of dihydromyricetin on the proliferation and migration of gastric cancer BGC-823 cells and explore the molecular mechanisms. METHODS: BGC-823 cells in routine culture were treated with different concentrations of dihydromyricetin (0, 40, 60, 80, 100, and 120 µg/mL) for 24 h, and the changes in cell viability were detected using CCK-8 assay; colony forming assay and Transwell assay were performed to assess the changes in colonyforming and migration abilities of the cells, respectively. The levels of MMP-2 and MMP-9 in the treated cells were determined using ELISA, and Western blotting was used to detect the expressions of E-cadherin, N-cadherin, cyclin D1, cyclin E1, HSP70 and HMGB1 and the phosphorylation levels of Akt and Stat3. RESULTS: CCK-8 assay showed that dihydromyricetin treatment dose-dependently inhibited the viability of BGC-823 cells (P < 0.05). Treatment with dihydromyricetin obviously suppressed the proliferation and migration of BGC-823 cells, significantly reduced the expression levels of cyclin D1, cyclin E1 and Ncadherin, enhanced E-cadherin expression, inhibited the phosphorylation of Akt and stat3, and downregulated HMGB1 expression in the cells. The results of ELISA demonstrated significantly lowered levels of MMP-2 and MMP-9 in dihydromyricetin-treated cells. CONCLUSIONS: Dihydromyricetin inhibits the proliferation and migration of BGC-823 cells through suppressing the activation of Akt/stat3 signaling pathways and HMGB1 expression.
Assuntos
Proteína HMGB1 , Neoplasias Gástricas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Flavonóis , Proteína HMGB1/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3RESUMO
Gastric cancer (GC) is a common malignancy tumour in China. Despite various therapeutic approaches to improve the survival rate of GC patients, the effectiveness of currently available treatments remains unsatisfactory. High mobility group box 1 (HMGB1) is reported to play a role in tumour development. However, the molecular mechanisms involved in HMGB1-mediated regulation of proliferation and migration of GC cells remain unclear. In the present study, we demonstrated that HMGB1 is highly expressed in GC cells and tissue. In HGC-27 GC cells, HMGB1 overexpression or HMGB1 RNA interference both demonstrated that HMGB1 could promote GC cell proliferation and migration. Investigation of the underlying molecular mechanisms revealed that HMGB1 enhanced cyclins expression, induced epithelial-to-mesenchymal transition and matrix metalloproteinase (MMPs) expression and promoted RAGE expression as well as RAGE-mediated activation of Akt/mTOR/P70S6K and ERK/P90RSK/CREB signalling pathways. We also found that inhibition of ERK and mTOR using specific inhibitors reduced recombinant human HMGB1-induced RAGE expression, suggesting that the RAGE-mTOR/ERK positive feedback loop is involved in HMGB1-induced GC cell proliferation and migration. Our study highlights a novel mechanism by which HMGB1 promotes GC cell proliferation and migration via RAGE-mediated Akt-mTOR and ERK-CREB signalling pathways which also involves the RAGE-mTOR/ERK feedback loop. These findings indicate that HMGB1 is a potential therapeutic target for GC.
RESUMO
BACKGROUND: Aloin has been reported to have many pharmacological effects including anti-inflammatory, anti-oxidant and anti-tumour activities. However, the precise molecular mechanisms underlying the anti-tumour properties of aloin are yet to be elucidated. METHODS: HGC-27 and BGC-823 gastric cancer cells were treated with aloin. EdU and colony formation assays were used to detect the proliferation ability of cells. The migration of cells was detected using wound healing and transwell assays. Western blotting was used to detect the levels of cyclinD1, cyclin E1, MMPs, N-cadherin, E-cadherin and NOX2. The phosphorylation of Akt, mTOR, P70S6K, S6, Src, stat3 and IκBα were also detected by Western blotting. Flow cytometry was used to detect the cell cycle distribution.The location of p65 in cells was determined by using a confocal microscopy assay. The total amounts of ROS present in cells were measured using an ROS assay kit. RESULTS: Here, we found that aloin inhibited the proliferation and migration of HGC-27 and BGC-823 gastric cancer cells using a combination of EdU, colony formation, wound healing and transwell assays. Further investigations revealed that aloin decreased the protein expression levels of cyclin D1, N-cadherin, and the matrix metalloproteinases (MMP)-2 and MMP-9; increased E-cadherin expression in a dose-dependent manner; inhibited reactive oxygen species (ROS) generation; and mediated the activation of Akt-mTOR, signal transducer and activator of transcription-3 (Stat3), and NF-κB signalling pathways. Our results also indicated that aloin is able to attenuate the expression levels of the two regulatory proteins of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), p47phox and p22phox, but had no effect on the level of gp91phox. N-acetylcysteine treatment of gastric cancer cells inhibited ROS production and Akt-mTOR, Stat3, and IκBα phosphorylation. Taken together, our data suggest that aloin inhibits the proliferation and migration of gastric cancer cells by downregulating NOX2-ROS-mediated activation of the Akt-mTOR, Stat3, and NF-κB signalling pathways. CONCLUSION: Our findings suggest a potential role for aloin in the prevention of gastric cancer cell proliferation and migration and provide novel insights into the anti-cancer properties of aloin.
Assuntos
Movimento Celular/efeitos dos fármacos , Emodina/análogos & derivados , NADPH Oxidase 2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Proliferação de Células/efeitos dos fármacos , Emodina/farmacologia , Humanos , Neoplasias Gástricas/metabolismo , Análise de Sobrevida , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism. METHODS: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 µg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 µg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy. RESULTS: Calenduloside E below 20 µg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1ß, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells. CONCLUSIONS: Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.
Assuntos
Transdução de Sinais , Animais , Lipopolissacarídeos , Camundongos , NF-kappa B , Ácido Oleanólico/análogos & derivados , Células RAW 264.7 , Espécies Reativas de Oxigênio , SaponinasRESUMO
Purpose: Aloin (ALO), a bioactive ingredient extracted from aloe vera, has anti-tumor effects. High Mobility Group Box 1 (HMGB1), a highly conserved nuclear DNA-binding protein, has been implicated in various cancer types. Highly expressed HMGB1 is closely associated with tumor cells apoptosis, proliferation and migration. We investigated the specific molecular mechanisms by which ALO-induced apoptosis by targeting HMGB1 in gastric cancer cells. Materials and methods: Human gastric cancer HGC-27 cells were treated with different doses of ALO (100, 200 and 400 µg/ml) for 24 h, after which DAPI staining was used to observe the nuclear morphology, Annexin V/PI double staining assay was used to determine the rate of apoptosis; Western blotting was used to detect the levels of PARP, pro-caspase3, HMGB1 and RAGE; nuclear translocation of HMGB1 was determined by conducting a nucleoplasm separation experiment. The Enzyme linked immunosorbent assay (ELISA) assay was used to detect release of HMGB1. The HGC-27 cells, transfected with HMGB1 shRNA plasmids, were stimulated with ALO for 24 h, after which a flow cytometry assay was used to detect the rate of apoptosis. HGC-27 cells were pre-treated with or without ALO and then stimulated with rhHMGB1, the phosphorylation of Akt, mTOR, P70S6K, S6, 4EBP1, ERK, P90RSK, cAMP regulatory element binding (CREB) were detected by Western blotting. Results: After different doses of ALO treatment, the nuclei showed morphological changes characteristic of apoptosis. Apoptotic rates were enhanced in a dose dependent manner. The level of cleaved PARP was enhanced and pro-caspase3, HMGB1 and RAGE levels were reduced, HMGB1 nuclear translocation and release were inhibited. The activation of rhHMGB1-induced Akt-mTOR-P70S6K and ERK-CREB signalling pathways was inhibited by ALO. Blocking these signalling pathways by special inhibitors and HMGB1 knockdown could enhance ALO-induced HGC-27 cell apoptosis. Conclusion: ALO- induced HGC-27 cell apoptosis by down-regulating expressions of HMGB1 and RAGE, inhibiting HMGB1 release and then suppressing rhHMGB1-induced activation of Akt-mTOR-P70S6K and ERK-P90RSK-CREB signalling pathways.
Assuntos
Apoptose/efeitos dos fármacos , Emodina/análogos & derivados , Proteína HMGB1/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Aloe/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Emodina/química , Emodina/farmacologia , Proteína HMGB1/metabolismo , Humanos , Estrutura Molecular , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
PURPOSE: Reactive oxygen species (ROS) are considered a direct cause of neurodegenerative diseases (NDDs). Drugs developed to target ROS are effective for the treatment of NDDs. Orientin is a pyrone glucoside extracted from Polygonum orientale, and it exhibits many pharmacological activities. In this study, we aimed to determine whether orientin could relieve hydrogen peroxide (H2O2)-induced neuronal apoptosis and to investigate the specific target of orientin. MATERIALS AND METHODS: In this study, the neuroprotective effect and its possible mechanisms of orientin in mouse pheochromocytoma cell line (PC12) cells stimulated by H2O2, establishing an oxidative stress model, were investigated. And we further tested the role of ROS in the neuroprotective effects of orientin. RESULTS: Orientin (5-100 µg/mL) did not cause toxicity in PC12 cells but significantly decreased H2O2-induced reduction in PC12 cell viability, cell apoptosis rates, and nuclear condensation. It also inhibited the activation of caspase-3 and degradation of poly(ADP-ribose) polymerase (PARP). Under the stimulation of H2O2, MAPKs (ERK, JNK, and p38), AKT, and Src signaling proteins in PC12 cells were activated in a time-dependent manner. The application of inhibitors that were specific for MAPKs, AKT, and Src effectively alleviated H2O2-induced cell apoptosis. In addition, the Src inhibitor decreased the activation of MAPKs and AKT signaling. More importantly, orientin effectively decreased H2O2-induced phosphorylation of MAPKs, AKT, and Src signaling proteins. Finally, we confirmed that orientin effectively inhibited H2O2-induced accumulation of ROS in cells. In addition, ROS inhibitors blocked the Src-MAPKs/AKT signaling pathway-dependent cell apoptosis stimulated by H2O2. CONCLUSION: These results indicate that alleviation of H2O2-induced cell apoptosis by orientin is Src-MAPKs/AKT dependent. Overall, our study confirms that orientin alleviates H2O2-induced cell apoptosis by inhibiting the ROS-mediated activation of Src-MAPKs/AKT signaling.
Assuntos
Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Glucosídeos/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/farmacologia , Conformação Molecular , Células PC12 , Ratos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: To investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. METHODS: RAW264.7 cells were treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100, 150, and 200 µg/mL) of chrysin for 24 h, and the cell viability was measured using CCK-8. RAW264.7 cells were pre-treated with 10, 30, or 60 µg/mL chrysin for 2 h before stimulation with LPS for different times. The levels of TNF-α, IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK- 1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe. The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy. RESULTS: Chrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells. At 10, 30, and 60 µg/mL, chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells; ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway. CONCLUSION: Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.
Assuntos
Anti-Inflamatórios/farmacologia , Flavonoides/farmacologia , Macrófagos/efeitos dos fármacos , Transdução de Sinais , Animais , Citocinas/metabolismo , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Salidroside, an active ingredient extracted from the Rhodiola rosea plant, has potential antitumor effects. However, the effects of salidroside on gastric cancer cell proliferation and migration remain unclear. In the present study, the inhibitory effects of salidroside on gastric cancer cell proliferation, migration and invasion and the molecular mechanisms underlying these effects were investigated. The human gastric cancer cell line, BGC823, was treated with different concentrations of salidroside (200, 400 and 600 µg/ml). Cell proliferation was determined with Cell Counting Kit8 and colony formation assays, and the migration and invasion of cells was detected by a wound healing and Transwell assay, respectively. Western blotting was performed to detect the levels of Ncadherin, Ecadherin and heat shock protein (HSP)70. In addition, the phosphorylation of protooncogene tyrosineprotein kinase Src (Src), protein kinase B (Akt), mitogen activated protein kinase 1 (ERK), signal transducer and activator of transcription (STAT)3 and focal adhesion kinase 1 (FAK) was examined by western blotting. The levels of matrix metalloproteinase (MMP)2 and MMP9 were determined by enzymelinked immunosorbent assay kits. Levels of reactive oxygen species (ROS) in cells were measured by a fluorescence plate reader with dichlorodihydrofluorescein diacetate. The results indicated that salidroside significantly suppressed cell proliferation and colony formation, inhibited cell migration and invasion, increased Ecadherin expression and decreased Ncadherin, MMP2 and MMP9 expression. Furthermore, salidroside suppressed ROS production and subsequently reduced the phosphorylation of Src, Akt, ERK and FAK. Salidroside also inhibited HSP70 expression, and HSP70 overexpression reversed the inhibitory effects of salidroside on BGC823 cell proliferation, migration and invasion. In conclusion, the present study revealed that salidroside inhibited the proliferation, migration and invasion of BGC823 cells by downregulating ROSmediated Srcassociated signaling pathway activation and HSP70 expression.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Neoplasias/metabolismo , Fenóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVE: To investigate the effect of aloin on apoptosis of human gastric cancer cells and explore the molecular mechanism. METHODS: Gastric cancer MKN-28 and HGC-27 cells were cultured routinely in 1640 medium supplemented with 10% fetal bovine serum and 10% non-essential amino acids (for HGC-27 cells) and treated with different concentrations of aloin for different durations. The cell viability, cell nuclear morphology, and apoptotic rate of the cells were detected using CCK-8 assay, DAPI staining and AnnexinV-FITC/PI, respectively; Western blotting was used to detect the expression levels of PARP, procaspase 3 and the phosphorylation of p38, ERK and JNK. The cells were treated with specific inhibitors of p38, ERK and JNK, and the inhibitory effects on these pathways were detected with Western blotting; DAPI staining was used to detect the effects of inhibitors on apoptosis of gastric cancer cells. RESULTS: Aloin dose-dependently inhibited the viability and induced apoptosis of HGC-27 and MKN-28 cells. Alion treatment obvious enhanced the phosphorylation of p38 and JNK but decreased ERK phosphorylation in the cells. Blocking ERK activation with the ERK inhibitor obviously enhanced aloin-induced cell apoptosis, where inhibiting p38 and JNK activation partly reversed alion-induced apoptosis in the cells. CONCLUSIONS: Aloin induces apoptosis of human gastric cancer cells in vitro by activating p38 and JNK signaling pathways and inhibiting ERK signaling pathway.
Assuntos
Apoptose/efeitos dos fármacos , Emodina/análogos & derivados , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Meios de Cultura , Relação Dose-Resposta a Droga , Emodina/farmacologia , Corantes Fluorescentes , Humanos , Indóis , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To study the effect of chrysin in inducing apoptosis of human hepatic carcinoma cells and explore the possible mechanism. METHODS: Human hepatic carcinoma SMMC-7721 cells treated with DMSO or chrysin at different concentrations (5-200 µg/mL) were examined for changes in the cell proliferation using CCK-8 assay. The morphological changes of SMMC-7721 cells were observed in response to treatment with 5, 10, or 20 µg/mL chrysin, and the changes in the cell nuclei were observed using DAPI nuclear staining. Annexin â ¤-FITC/PI flow cytometry was used to determine the cell apoptosis rate. The changes in the apoptosis-related proteins (PARP and caspase-3) and MAPKs signal pathway were detected with Western blotting. RESULTS: Chrysin treatment obviously suppressed the proliferation of SMMC-7721 cells in a dose-dependent manner below the concentration of 60 µg/mL. Chrysin (20 µg/mL) also caused significantly increased cell apoptosis and significant cleavage of PARP and caspase-3. Chrysin significantly activated MAPKs signaling pathway in a time-and dose-dependent manner, with the peak activation level occurring at 15 min. Pretreatment of the cells with specific inhibitors of the MAPKs pathway obviously inhibited the effect of chrysin in inducing cell apoptosis. CONCLUSIONS: Chrysin inhibits the proliferation and promotes apoptosis of SMMC-7721 cells by regulating the activation of MAPKs signaling.
Assuntos
Apoptose , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Flavonoides/farmacologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Dimetil Sulfóxido , Relação Dose-Resposta a Droga , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Transdução de SinaisRESUMO
Myricitrin, a naturally occurring polyphenol hydroxy flavonoid, has been reported to possess anti-inflammatory properties. However, the precise molecular mechanism of myricitrin's effects on LPS-induced inflammation is unclear. In the present study, myricitrin significantly alleviated acute lung injury in mice. Myricitrin also markedly suppressed the production of NO, TNF-α, IL-6, and MCP-1 in RAW264.7 macrophage cells. The inhibition of NO was concomitant with a decrease in the protein and mRNA levels of iNOS. The phosphorylation of JAKs and STAT-1 was abrogated by myricitrin. Furthermore, myricitrin inhibited the nuclear transfer and DNA binding activity of STAT1. The JAK-specific inhibitor ruxolitinib simulated the anti-inflammatory effect of myricitrin. However, myricitrin had no impact on the MAPK signalling pathway. Myricitrin attenuated the generation of intracellular ROS by inhibiting the assembly of components of the gp91phox and p47phox. Suppression of ROS generation using NAC or apocynin or by silencing gp91phox and p47phox all demonstrated that decreasing the level of ROS inhibited the LPS-induced inflammatory response. Collectively, these results confirmed that myricitrin exhibited anti-inflammatory activity by blocking the activation of JAKs and the downstream transcription factor STAT1, which may result from the downregulation of NOX2-dependent ROS production mediated by myricitrin.
Assuntos
Flavonoides/farmacologia , Janus Quinases/metabolismo , Lipopolissacarídeos/toxicidade , NADPH Oxidase 2/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Camundongos , Células RAW 264.7RESUMO
HMGB1, a highly conserved nonhistone DNA-binding protein, plays an important role in inflammatory diseases. Once released to the extracellular space, HMGB1 acts as a proinflammatory cytokine that triggers inflammatory reaction. Our previous study showed that salidroside exerts anti-inflammatory effect via inhibiting the JAK2-STAT3 signalling pathway. However, whether salidroside inhibits the release of HMGB1 is still unclear. In this study, we aim to study the effects of salidroside on HMGB1 release and then investigate the potential molecular mechanisms. In an experimental rat model of sepsis caused by CLP, salidroside administration significantly attenuated lung injury and reduced the serum HMGB1 level. In RAW264.7 cells, we investigated the effects of salidroside on LPS-induced HMGB1 release and then explored the underlying molecular mechanisms. We found that salidroside significantly inhibited LPS-induced HMGB1 release, and the inhibitory effect was correlated with the HMGB1 acetylation levels. Mechanismly, salidroside inhibits HMGB1 acetylation through the AMPK-SirT1 pathway. In addition, SirT1 overexpression attenuated LPS-induced HMGB1 acetylation and nucleocytoplasmic translocation. Furthermore, in SirT1 shRNA plasmid-transfected cells, salidroside treatment enhanced SirT1 expression and reduced LPS-activated HMGB1 acetylation and nucleocytoplasmic translocation. Collectively, these results demonstrated that salidroside might reduce HMGB1 release through the AMPK-SirT1 signalling pathway and suppress HMGB1 acetylation and nucleocytoplasmic translocation.