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Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that there appeared to be two instances of overlapping data panels comparing between the cell migration and invasion assay data shown in Figs. 4 and 6 on p. 143 and 145, respectively, such that data which were intended to represent the results from differently performed experiments had apparently been derived from the same original sources. In addition, the authors themselves realized that incorrect western blotting data for Snail protein in Fig. 10A on p. 147 had been included in the figure. The authors were able to reexamine their original data files, and realized that the affected data panels in these figures had inadvertently been incorporated into them incorrectly. The revised versions of Figs. 4, 6, and 10, featuring the correct data for the 'NC / Control' panels in Fig. 4B and C and the 'siRNA2 / ATP 12 h' panels in Fig. 4A and B, a replacement data panel for the 'siRNA1 / Control' experiment in Fig. 6, and the correct western blotting data for Snail protein in Fig. 10A (together with a revised histogram for the MCF7 cell line relating to Fig. 10A) are shown on the next three pages. The authors wish to emphasize that the errors made in compiling these figures did not affect the overall conclusions reported in the paper, and they are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this corrigendum. All the authors agree to the publication of this corrigendum, and also apologize to the readership for any inconvenience caused. [Oncology Reports 39: 138150, 2018; DOI: 10.3892/or.2017.6081].
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The pathophysiology of autism spectrum disorders (ASDs) is causally linked to postsynaptic scaffolding proteins, as evidenced by numerous large-scale genomic studies [1, 2] and in vitro and in vivo neurobiological studies of mutations in animal models [3, 4]. However, due to the distinct phenotypic and genetic heterogeneity observed in ASD patients, individual mutation genes account for only a small proportion (<2%) of cases [1, 5]. Recently, a human genetic study revealed a correlation between de novo variants in FERM domain-containing-5 (FRMD5) and neurodevelopmental abnormalities [6]. In this study, we demonstrate that deficiency of the scaffolding protein FRMD5 leads to neurodevelopmental dysfunction and ASD-like behavior in mice. FRMD5 deficiency results in morphological abnormalities in neurons and synaptic dysfunction in mice. Frmd5-deficient mice display learning and memory dysfunction, impaired social function, and increased repetitive stereotyped behavior. Mechanistically, tandem mass tag (TMT)-labeled quantitative proteomics revealed that FRMD5 deletion affects the distribution of synaptic proteins involved in the pathological process of ASD. Collectively, our findings delineate the critical role of FRMD5 in neurodevelopment and ASD pathophysiology, suggesting potential therapeutic implications for the treatment of ASD.
Assuntos
Transtorno do Espectro Autista , Modelos Animais de Doenças , Proteínas de Membrana , Transtornos do Neurodesenvolvimento , Animais , Camundongos , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Masculino , Neurônios/metabolismo , Comportamento Animal/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Camundongos Knockout , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Camundongos Endogâmicos C57BL , Comportamento Social , Comportamento Estereotipado , Sinapses/metabolismo , FemininoRESUMO
Innate defensive behavior is important for animal survival. The Vglut2+ neurons in the ventral tegmental area (VTA) have been demonstrated to play important roles in innate defensive behaviors, but the neural circuit mechanism is still unclear. Here, we find that VTA - zona incerta (ZI) glutamatergic projection is involved in regulating innate fear responses. Combining calcium signal recording and chemogentics, we find that VTA-Vglut2+ neurons respond to foot shock stimulus. Inhibition of VTA-Vglut2+ neurons reduces foot shock-evoked freezing, while chemogentic activation of these neurons results in an enhanced fear response. Using viral tracing and immunofluorescence, we show that VTA - Vglut2+ neurons send direct excitatory outputs to the ZI. Moreover, we find that the activity of VTAVglut2 - ZI projection is pivotal in modulating fear response. Together, our study reveals a new VTA - ZI glutamatergic circuit in mediating innate fear response and provides a potential target for treating post-traumatic stress disorder.
Assuntos
Área Tegmentar Ventral , Zona Incerta , Animais , Área Tegmentar Ventral/fisiologia , Neurônios/fisiologia , Imunofluorescência , Medo/fisiologiaRESUMO
We have previously demonstrated that extracellular adenosine 5'-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.
Assuntos
Neoplasias da Mama , Trifosfato de Adenosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.
Assuntos
Proteínas de Transporte/metabolismo , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Proteínas dos Microfilamentos/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Actinas , Humanos , Lisina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
C-type lectins are carbohydrate-binding proteins that play important roles in immunity by serving as pattern recognition receptors. In the present study, a novel nattectin-like C-type lectin was obtained from the weather loach, Misgurnus anguillicaudatus, designated as MaCTL. MaCTL encodes a peptide with 165 amino acids, with a signal peptide and a single C-type lectin domain (CTLD), containing a galactose-specific QPD motif and a conserved Ca2+ -binding site. Transcripts of MaCTL were significantly upregulated after immune challenge with its pathogen A. hydrophila. In vitro assays with recombinant MaCTL protein revealed that it exhibited hemagglutinating and bacterial agglutinating activities, in a Ca2+ -dependent manner. MaCTL was found to bind to a wide range of bacteria, as well as bind to bacterial polysaccharides LPS and PGN. Moreover, MaCTL displayed antimicrobial activity by inhibiting the growth of bacteria. These results collectively suggest that MaCTL is involved in the antibacterial defence of weather loach.
Assuntos
Cipriniformes/imunologia , Doenças dos Peixes/imunologia , Lectinas Tipo C/imunologia , Aeromonas hydrophila/efeitos dos fármacos , Aglutinação , Sequência de Aminoácidos , Animais , Cipriniformes/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/imunologia , Lectinas Tipo C/química , Lectinas Tipo C/genética , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
Our previous research demonstrated that extracellular adenosine 5'-triphosphate (ATP) could promote breast cancer cell invasion. However, the impact of extracellular ATP on chemoresistance and the mechanisms behind ATP pro-invasion and pro-chemoresistance remain unclear. Here we aimed to determine the molecules or signaling pathways involved. cDNA microarray was performed to identify the differentially expressed genes before and after ATP treatment. As a result, Sex-determining region Y-box 9 (SOX9) was up-regulated after ATP treatment in breast cancer cells. In vitro invasion and migration assays demonstrated that knocking down SOX9 attenuated ATP-driven invasive capability. Mass spectrometry and co-IP revealed that SOX9 interacted with Janus kinase 1 (JAK1). Afterward, IL-6-JAK1-STAT3 signaling was demonstrated to promote SOX9 expression and invasion following ATP treatment. Notably, ATP-IL-6-SOX9 signaling was shown to stimulate chemoresistance in breast cancer cells. ChIP assays identified some potential SOX9 target genes, among which carcinoembryonic antigen-related cell adhesion molecule 5/6 (CEACAM5/6) was demonstrated to mediate ATP pro-invasive function, while ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) mediated ATP-driven chemoresistance. In addition, SOX9-knockdown and apyrase (an ATP hydrolase)-treated MDA-MB-231 cells illustrated decreased tumor growth and enhanced drug sensitivity in nude mice. In vitro spheroid formation assays also proved the significance of ATP-SOX9 in mediating chemoresistance. Moreover, molecules involved in ATP-SOX9 signaling were up-regulated in human breast carcinoma specimens and were associated with poor prognosis. Altogether, SOX9 signaling is vital in ATP-driven invasion and chemoresistance, which may serve as a potential target for breast cancer therapies.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Fatores de Transcrição SOX9/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Transdução de Sinais , TransfecçãoRESUMO
Extracellular ATP has been shown to play an important role in invasion and the epithelial-mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia-inducible factor (HIF) signaling and upregulate hypoxia-inducible factor 1/2α (HIF-1/2α) expression. After knocking down HIF-1/2α using siRNA, we found that ATP-driven invasion and EMT were significantly attenuated via HIF2A-siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF-2α direct targets, among which lysyl oxidase-like 2 (LOXL2) and matrix metalloproteinase-9 (MMP-9) mediated ATP-driven invasion, and E-cadherin and Snail mediated ATP-driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF-2α and mediate ATP-driven HIF-2α upregulation. Furthermore, we demonstrated that expressions of HIF-2α and its target proteins could be regulated via ATP by AKT-PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF-2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP-HIF-2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF-2α signaling, which may be a potential target for future anti-metastasis therapy.
Assuntos
Trifosfato de Adenosina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Hipóxia/patologia , Invasividade Neoplásica/patologia , Aminoácido Oxirredutases/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular/efeitos dos fármacos , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Animais , Apirase/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Niclosamida/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Regulação para CimaRESUMO
Adenosine 5'-triphosphate (ATP) is one of the most abundant biochemical constituents within the tumor microenvironment and is postulated to play critical roles in the progression of a number of types of tumors via interaction with the P2Y2 receptor. In the present study, we demonstrated that the P2Y2 receptor was highly expressed in MCF7 and Hs578T breast cancer cells. Downregulation of the P2Y2 receptor by small interfering RNA (siRNA) significantly attenuated ATP- or UTP-driven migration and invasion of the breast cancer cells as well as expression of EMT-related genes Snail and E-cadherin. Consistent with the observations in vitro, the P2Y2 receptor was found to be abundantly expressed at the invasive edge of the tumor, in infiltrating tumor cells in breast adipose tissues and/or the cancer embolus in the lymphatic sinuses compared with the tumor core areas. Furthermore, high Snail expression and weak or negative expression of E-cadherin were observed at the invasive edge of tumors. Taken together, these data indicate that the P2Y2 receptor promoted cell migration and invasion in breast cancer cells via EMT-related genes Snail and E-cadherin.
Assuntos
Neoplasias da Mama/patologia , Caderinas/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Adulto , Idoso , Antígenos CD , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Fatores de Transcrição da Família Snail/genética , Regulação para Cima , Adulto JovemRESUMO
Extracellular adenosine 5'-triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro-invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of ß-catenin as well as its downstream target genes CD44, c-Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP-driven events in vitro and in vivo. Furthermore, iCRT14, a ß-catenin/TCF complex inhibitor, could also suppress ATP-driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2-ß-catenin axis. Thus blockade of the ATP-P2Y2-ß-catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/patologia , Invasividade Neoplásica/patologia , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase em Tempo RealRESUMO
As one member of G protein-coupled P2Y receptors, P2Y2 receptor can be equally activated by extracellular ATP and UTP. Our previous studies have proved that activation of P2Y2 receptor by extracellular ATP could promote prostate cancer cell invasion and metastasis in vitro and in vivo via regulating the expressions of some epithelial-mesenchymal transition/invasion-related genes (including IL-8, E-cadherin, Snail and Claudin-1), and the most significant change in expression of IL-8 was observed after P2Y2 receptor activation. However, the signaling pathway downstream of P2Y2 receptor and the role of IL-8 in P2Y2-mediated prostate cancer cell invasion remain unclear. Here, we found that extracellular ATP/UTP induced activation of EGFR and ERK1/2. After knockdown of P2Y2 receptor, the ATP -stimulated phosphorylation of EGFR and ERK1/2 was significantly suppressed. Further experiments showed that inactivation of EGFR and ERK1/2 attenuated ATP-induced invasion and migration, and suppressed ATP-mediated IL-8 production. In addition, knockdown of IL-8 inhibited ATP-mediated invasion and migration of prostate cancer cells. These findings suggest that P2Y2 receptor and EGFR cooperate to upregulate IL-8 production via ERK1/2 pathway, thereby promoting prostate cancer cell invasion and migration. Thus blocking of the P2Y2-EGFR-ERK1/2 pathway may provide effective therapeutic interventions for prostate cancer.
Assuntos
Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Próstata/metabolismo , Receptores Purinérgicos P2Y2/genética , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Interleucina-8/agonistas , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Cross-Talk , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais , Uridina Trifosfato/metabolismo , Uridina Trifosfato/farmacologiaRESUMO
OBJECTIVE: To isolate P-cadherin positive and negative oral gingival epithelial cells, and to compare the biological characteristics with junctional epithelial cells. METHODS: Human oral gingival epithelial cells and junctional epithelial cells were cultured. P-cadherin positive and negative cells were isolated from oral gingival epithelial cells. The cellular adhesion, proliferation and migration were measured and compared. RESULTS: The P-cadherin positive cells accounted for 20% of oral gingival epithelial cells. Compared with juctional epithelial cells, P-cadherin positive oral gingival epithelial cells showed similar properties of adhesion and migration, and stronger proliferation ability (0.72 ± 0.06 vs. 0.60 ± 0.05, P<0.05). P-cadherin negative oral gingival epithelial cells showed weaker ability of adhesion (48% ± 6% vs. 87% ± 11%, P<0.05), proliferation (0.36 ± 0.04 vs. 0.60 ± 0.05, P<0.05) and migration (10.3 ± 2.7 vs. 23.4 ± 4.8, P<0.05). CONCLUSION: P-cadherin positive oral gingival epithelial cells showed some similar but different biological characteristics, compared with juctional epithelial cells, which suggested that during the process of transforming oral gingival epithelial cells into juctional epithelial cells, complex gene and protein changes were involved instead of simply cellular migration.
Assuntos
Células Epiteliais/citologia , Gengiva/citologia , Caderinas , Adesão Celular , Diferenciação Celular , Movimento Celular , Células Cultivadas , HumanosRESUMO
The ATP-gated P2X7 has been shown to play an important role in invasiveness and metastasis of some tumors. However, the possible links and underlying mechanisms between P2X7 and prostate cancer have not been elucidated. Here, we demonstrated that P2X7 was highly expressed in some prostate cancer cells. Down-regulation of P2X7 by siRNA significantly attenuated ATP- or BzATP-driven migration and invasion of prostate cancer cells in vitro, and inhibited tumor invasiveness and metastases in nude mice. In addition, silencing of P2X7 remarkably attenuated ATP- or BzATP- driven expression changes of EMT/invasion-related genes Snail, E-cadherin, Claudin-1, IL-8 and MMP-3, and weakened the phosphorylation of PI3K/AKT and ERK1/2 in vitro. Similar effects were observed in nude mice. These data indicate that P2X7 stimulates cell invasion and metastasis in prostate cancer cells via some EMT/invasion-related genes, as well as PI3K/AKT and ERK1/2 signaling pathways. P2X7 could be a promising therapeutic target for prostate cancer.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Purinérgicos P2X7/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMO
BACKGROUND: Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug. METHODS: The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice. RESULTS: AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G0/G1 cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger - N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10-40mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index. CONCLUSIONS AND GENERAL SIGNIFICANCE: These data support development of AD-1 as a potential agent for lung cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Ginsenosídeos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , CamundongosRESUMO
OBJECTIVE: To investigate the effects of Qushuanling Capsule ( QSLC) on thrombus formation and platelet aggregation in rats. METHODS: Arteriovenous bypass, venous thrombosis, and middle cerebral artery thrombosis models were used in rats to investigate the anti-thrombotic effects of QSLC, a compound of nine Chinese herbs. The platelet aggregation induced by adenosine diphosphate (ADP), thrombin or arachidonic acid (AA), as well as the contents of thromboxane B(2) (TXB(2)) and 6-keto-prostaglandin F1α (6-keto-PGF1α) in rat plasma and aortic walls, were determined to investigate the possible mechanisms of the anti-thrombotic effects of QSLC. RESULTS: After oral administration with QSLC for 7 days, arteriovenous bypass thrombosis was obviously suppressed compared with the model group, venous thrombosis was also obviously suppressed, rat behaviors were obviously improved, and brain infarct size as well as water content were also reduced. The platelet aggregation induced by ADP or thrombin was inhibited by QSLC, but the drug had no effect on AA-induced platelet aggregation and content of TXB(2) and 6-keto-PGF1α in plasma and the aortic wall. CONCLUSION: These results suggest that QSLC can be used in the prevention and treatment of thrombotic diseases, and that its mechanism of action may be related to inhibition of platelet aggregation.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Trombose/tratamento farmacológico , Trombose/patologia , 6-Cetoprostaglandina F1 alfa/sangue , Difosfato de Adenosina/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Infarto Cerebral/sangue , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/patologia , Masculino , Artéria Cerebral Média/efeitos dos fármacos , Artéria Cerebral Média/patologia , Ratos , Ratos Sprague-Dawley , Tromboxano B2/sangue , Trombose Venosa/tratamento farmacológico , Trombose Venosa/patologiaRESUMO
We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-ß1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-ß1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-ß1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-ß1 and TIEG1.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Medula Óssea/metabolismo , Cistanche/química , Expressão Gênica/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Extratos Vegetais/farmacologia , Fosfatase Ácida/sangue , Animais , Conservadores da Densidade Óssea/isolamento & purificação , Medula Óssea/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Estradiol/sangue , Feminino , Fêmur/efeitos dos fármacos , Fêmur/patologia , Hormônio Foliculoestimulante/sangue , Humanos , Isoenzimas/sangue , Hormônio Luteinizante/sangue , Osteocalcina/sangue , Osteoporose Pós-Menopausa/sangue , Ovariectomia , Extratos Vegetais/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ratos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Solventes/química , Fosfatase Ácida Resistente a Tartarato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Água/químicaRESUMO
OBJECTIVE: To investigate the effects of puerarin on the activity of superoxide dismutase (SOD), and expressions of advanced glycation end-product (AGE) receptor (RAGE) and vascular endothelial growth factor (VEGF) in retinas of streptozotocin (STZ)-induced early diabetic rats. METHODS: Diabetic rat models were established by inducing diabetes via intra-peritoneal injection of STZ. Rats were randomly divided into normal (control), diabetic (DM), and DM+ puerarin groups. After intra-gastric administration of puerarin (500 mg/kg/day for 4 weeks), levels of SOD and malondialdehyde (MDA) were determined in serum and retina. mRNA and protein expression levels of RAGE and VEGF in retinas were determined by real-time polymerase chain reaction (RT-PCR) (mRNA) and Western blot analysis (protein levels). RESULTS: There was significantly lower SOD activity and significantly higher MDA in serum and retinas of the DM group compared with the two other groups (P<0.05). After treatment with puerarin, SOD activity increased and MDA content decreased in this group (P<0.05). mRNA and protein expression levels of RAGE and VEGF in the DM group were significantly higher than those of the other groups (P<0.05), and decreased after puerarin treatment (P<0.05). CONCLUSIONS: Puerarin is able to enhance SOD activity, and inhibit RAGE and VEGF expressions in retinas of STZ-induced early diabetic rats.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Ativadores de Enzimas/administração & dosagem , Isoflavonas/administração & dosagem , Receptores Imunológicos/antagonistas & inibidores , Retina/patologia , Superóxido Dismutase/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Western Blotting , Diabetes Mellitus Experimental/patologia , Perfilação da Expressão Gênica , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada , Resultado do TratamentoRESUMO
Gestational trophoblastic disease (GTD) includes frankly malignant choriocarcinoma (CCA) and placental site trophoblastic tumor and potentially malignant hydatidiform mole. p21-Activated kinase (PAK) 4 promotes cell motility. This study investigated the role of PAK4 in the pathogenesis of GTD. PAK4 messenger RNA and protein expressions in clinical samples and cell lines of normal placentas and GTD were determined by quantitative real-time polymerase chain reaction and western blot, respectively. The effects of human chorionic gonadotropin (hCG) and phosphoinositide 3 kinase (PI3K) on the expression and activation of PAK4 were investigated by treating CCA JEG3 and JAR cells with anti-hCG antibody and PI3K inhibitor, respectively. The effects of PAK4 on CCA cell proliferation, migration and invasion were assessed by corresponding functional assays. We demonstrated overexpression of PAK4 in GTD and CCA cell lines at both RNA and protein level. hCG is one of the upstream regulators of PAK4 expression, whereas activation of PAK4 is PI3K/PKB dependent in JEG3 and JAR cells. Significant correlation was found between PAK4 expression and proliferation index minichromosome maintenance complex component 7 (P = 0.007). In JEG3 and JAR cells, stably transfected PAK4 increased proliferation, migration and invasion, whereas small interfering RNA knockdown of PAK4 decreased proliferation, migration and invasion along with downregulated CDK6 and membrane-type 1 matrix metalloproteinase (MT1-MMP) and upregulated p16. We further found PAK4-mediated transcription of MT1-MMP in CCA cells by luciferase reporter assay. Our results demonstrated for the first time that overexpressed PAK4 was involved in the pathogenesis of GTD, promoting proliferation and enhancing cell migration and invasion in CCA cells.
Assuntos
Movimento Celular , Proliferação de Células , Coriocarcinoma/patologia , Neoplasias Uterinas/patologia , Quinases Ativadas por p21/metabolismo , 1-Fosfatidilinositol 4-Quinase/farmacologia , Apoptose , Western Blotting , Adesão Celular , Linhagem Celular Tumoral , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Gonadotropina Coriônica/farmacologia , Feminino , Doença Trofoblástica Gestacional , Humanos , Técnicas Imunoenzimáticas , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Trofoblásticas/genética , Neoplasias Trofoblásticas/metabolismo , Neoplasias Trofoblásticas/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genéticaRESUMO
Using a Dual-View wavelength splitter, double-stained African green monkey kidney COS-7 cells, transfected with pEGFP-Myosin 15a and costained with Rhodamine-filopodia were observed based on an ICCD(intensified charge couple device) fluorescence micro-imaging systems. Total internal reflection fluorescence microscopy was used to observe the overexpression of Myosin 15a to the tips of the elongation filopodia. An approach to collecting fluorescence in two channels and avoiding spectra crosstalk was employed to observe Myosin 15a and filopodia distribution in African green monkey kidney COS-7 cells. High sensitivity TIRF technology and two channels imaging method provided a wide application in bio-medical studies.