RESUMO
Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.
Assuntos
Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Proteínas RGS/genética , Ribonucleases/genética , Fatores de Transcrição/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Immunoblotting , Leupeptinas/farmacologia , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteínas RGS/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismoRESUMO
Lung cancer is still the number one cause of death from cancer worldwide. The clinical effect of platinum-based chemotherapy for non-small cell lung cancer is constrained by the resistance to drug. To overcome chemo-resistance, various modified treatment including combination therapy has been used, but overall survival has not been improved yet. In this study, chemo-resistant lung cancer cells, A549/Cis and H460/Cis, were developed by long-term exposure of cells to cisplatin and the proliferative capability of these resistant cells was verified to be reduced. We found cytotoxic effect of epigallocatechin gallate (EGCG), a major catechin derived from green tea, on both the parental lung cancer cells, A549 and H460, and their cisplatin resistant cells, A549/Cis and H460/Cis. ELISA and Western blot analysis revealed that EGCG was able to increase interlukine-6 (IL-6) production per cell, whereas its downstream effector Signal transducers and activators of transcription 3 (STAT3) phosphorylation was not changed by EGCG, indicating that IL-6/STAT3 axis is not the critical signaling to be inhibited by EGCG. We next found that EGCG suppresses the expression of both Axl and Tyro 3 receptor tyrosine kinases at mRNA and protein level, explaining the cytotoxic effect of EGCG on lung cancer cells, especially, regardless of cisplatin resistance. Taken together, these data suggest that EGCG impedes proliferation of lung cancer cells including their chemo-resistant variants through downregulation of Axl and Tyro 3 expression.
RESUMO
BACKGROUND: Laminar flow protects from atherosclerosis in endothelium. RESULTS: Laminar flow induces Nrf2 activation dependent on ERK5 activation, leading to up-regulation of downstream genes of Nrf2. CONCLUSION: ERK5 requires Nrf2 activation to exert cytoprotective effect on HUVEC. ERK5 inhibitor BIX02189 regulates Nrf2 activation in vivo. SIGNIFICANCE: Identifying ERK5 as a molecular target for regulating flow-mediating Nrf2-dependent gene expression may have significant therapeutic potential for treating atherosclerosis. Atherosclerosis is often observed in areas where disturbed flow is formed, whereas atheroprotective region is found in areas where steady laminar flow is developed. It has been reported that some genes activated by blood flow play important roles in vascular function and pathogenesis of atherosclerosis. Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction and disease under laminar flow. Krüppel-like factor 2 (KLF2) and NF-E2-related factor 2 (Nrf2) are major transcriptional factors that contribute to anti-atherogenic responses under laminar flow. Implication of ERK5 in laminar flow-mediated regulation of KLF2-dependent gene has been established, whereas the role of ERK5 in laminar flow-mediated activation of Nrf2 pathway has not been addressed yet. In this study, we found that the blockage of ERK5 either by genetic depletion with siRNA or by biochemical inactivation with a specific chemical compound inhibited laminar flow-induced up-regulation of Nrf2-dependent gene expressions, whereas activation of ERK5 increased transcriptional activity and nuclear translocation of Nrf2, which suggests that ERK5 mediates laminar flow-induced up-regulation of Nrf2-dependent gene expression. Further functional studies showed that ERK5 provides protection against oxidative stress-induced cytotoxicity dependent on Nrf2. Molecular interaction between ERK5 and Nrf2 was further induced by laminar flow. Finally, flow-dependent nuclear localization of Nrf2 was inhibited by BIX02189, a specific inhibitor of MEK5, in aorta of mice in vivo. Collectively, these data demonstrate that laminar flow-induced activation of ERK5-Nrf2 signal pathway plays a critical role for anti-inflammatory and anti-apoptotic mechanism in endothelial cells.
Assuntos
Aterosclerose/prevenção & controle , Aterosclerose/fisiopatologia , Endotélio Vascular/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fluxo Sanguíneo Regional , Ativação Transcricional , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 7 Ativada por Mitógeno/genética , Fator 2 Relacionado a NF-E2/metabolismo , Regulação para CimaRESUMO
Perfluorohexane sulfonate (PFHxS) is a widely detected replacement for legacy long-chain perfluoroalkyl substances (PFAS) in the environment and human blood samples. Its potential toxicity led to its recent classification as a globally regulated persistent organic pollutant. Although animal studies have shown a positive association between PFHxS levels and hepatic steatosis and hepatocellular hypertrophy, the link with liver toxicity, including end-stage liver cancer, remains inconclusive. In this study, we examined the effects of PFHxS on the proliferation of Hep3B (human hepatocellular carcinoma) and SK-Hep1 (human liver sinusoidal endothelial cells). Cells were exposed to different PFHxS concentrations for 24-48 h to assess viability and 12-14 days to measure colony formation. The viability of both cell lines increased at PFHxS concentrations <200 µM, decreased at >400 µM, and was highest at 50 µM. Colony formation increased at <300 µM and decreased at 500 µM PFHxS. Consistent with the effect on cell proliferation, PFHxS increased the expression of proliferating cell nuclear antigen (PCNA) and cell-cycle molecules (CDK2, CDK4, cyclin E, and cyclin D1). In summary, PFHxS exhibited a biphasic effect on liver cell proliferation, promoting survival and proliferation at lower concentrations and being cytotoxic at higher concentrations. This suggests that PFHxS, especially at lower concentrations, might be associated with HCC development and progression.
Assuntos
Ácidos Alcanossulfônicos , Carcinoma Hepatocelular , Poluentes Ambientais , Fluorocarbonos , Neoplasias Hepáticas , Animais , Humanos , Ácidos Sulfônicos , Células Endoteliais , Neoplasias Hepáticas/induzido quimicamente , Alcanossulfonatos , Fluorocarbonos/toxicidade , Proliferação de Células , Ácidos Alcanossulfônicos/toxicidadeRESUMO
The effect of agonists on AMP-activated protein kinase (AMPK), mainly metformin and phenformin, has been appreciated in the treatment of multiple types of tumors. Specifically, the antitumor activity of phenformin has been demonstrated in melanomas containing the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation. In this report, we elucidated the synergistic antitumor effects of biguanides with metabolism inhibitors on colon tumors. Phenformin with 2-deoxy-D-glucose (2DG) inhibited tumor cell growth in cancer cell lines, including HT29 cells harboring BRAF- and p53-mutations. Biochemical analyses showed that two chemotherapeutics exerted cooperative effects to reduce tumor growth through cell cycle arrest, apoptosis, and autophagy. The drugs demonstrated activity against phosphorylated ERK and the gain-of-function p53 mutant protein. To demonstrate tumor regressive effects in vivo, we established patient-derived models, including xenograft (PDX) and organoids (PDO). Co-treatment of biguanides with chemotherapeutics efficiently reduced the growth of patient-derived colon models in comparison to treatment with a single agent. These results strongly suggest that significant therapeutic advantages would be achieved by combining AMPK activators such as phenformin and cancer metabolic inhibitors such as 2DG.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Metformina , Animais , Camundongos , Humanos , Fenformin/farmacologia , Fenformin/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteína Supressora de Tumor p53 , Proteínas Quinases Ativadas por AMP/metabolismo , Reposicionamento de Medicamentos , Neoplasias do Colo/tratamento farmacológico , Metformina/farmacologia , Metformina/uso terapêuticoRESUMO
Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a calcium-independent phospholipase A(2) (iPLA(2))-dependent manner. The iPLA(2)beta-specific inhibitor S-BEL or iPLA(2)beta small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA(2)beta by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA(2)beta-Akt signaling pathway, and control ROS production and foam cell formation.
Assuntos
Células Espumosas/imunologia , Regulação da Expressão Gênica/imunologia , Fosfolipases A2 do Grupo IV/imunologia , NADH NADPH Oxirredutases/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Animais , Aterosclerose/imunologia , Aterosclerose/metabolismo , Western Blotting , Sinalização do Cálcio/imunologia , Linhagem Celular , Citometria de Fluxo , Células Espumosas/metabolismo , Expressão Gênica , Fosfolipases A2 do Grupo IV/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Microscopia Confocal , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
Non-small-cell lung cancer (NSCLC) is the most frequent cause of cancer-related death. In this study, we found the anticancer activity of lotus seedpod extract (LSPE) in NSCLC cells, since LSPE treatment inhibited cell proliferation of A549 and H460 cells in a dose-dependent manner and the clonogenic activities of LSPE-treated cells were also reduced. In LSPE-treated cells, the cleavage of poly (ADP-ribose) polymerase (PARP) and phosphorylation of H2X, were also observed, indicating the pro-apoptotic effect of LSPE. Next, we found that LPSE treatment diminished the levels of protein and mRNA of Axl, a receptor tyrosine kinase (RTK) that transduces critical signals for cell proliferation and inhibition of apoptosis. The promoter activity of Axl was found to be dose-dependently decreased in response to LSPE treatment, implying that LSPE inhibited Axl gene expression at transcriptional level. In addition, Axl overexpression was found to decrease the effects of LSPE on inhibition of cell proliferation and colony formation as well as induction of PARP cleavage and phosphorylation of H2AX, while the same activities of LPSE were increased by knockdown of Axl gene expression, indicating that the antiproliferative and pro-apoptotic effect of LSPE is inversely proportional to the protein level of Axl. Taken together, we found that the LSPE suppressed cell proliferation and induced apoptosis of NSCLC cells, which is attenuated or augmented by overexpression or RNA interference of Axl expression, respectively. Our data suggest that Axl is a novel therapeutic target of LSPE to inhibit cell proliferation and promote apoptosis in NSCLC cells. PRACTICAL APPLICATIONS: In this study, lotus seedpod extract (LSPE) was found to have the cytotoxic and apoptosis-inducing potentials in non-small-cell lung cancer (NSCLC) cells. LSPE downregulated the Axl expression at transcriptional level and the effects of LSPE on cell proliferation as well as apoptosis were affected by Axl protein level. Therefore, the inference of Axl-mediated intracellular signals by LSPE must be a novel approach to control NSCLC. Since our data imply that LSPE contains bioactive compounds targeting Axl, further studies to elucidate these compounds might discover a potent therapeutic agent.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Lotus , Neoplasias Pulmonares , Nelumbo , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Neoplasias Pulmonares/tratamento farmacológico , Extratos Vegetais/farmacologia , SementesRESUMO
Acrylamide is known as a neurotoxicant found in commonly consumed food as well as in human body. However, the underlying mechanisms involved in neurotoxicity by acrylamide and its metabolite, glycidamide remain largely unknown. In this study, we have examined the interplay between CYP2E1, AMPK, ERK and PKC in acrylamide-induced neurotoxicity associated with autophagy in PC12 cells. Acrylamide-induced cell death was mediated by CYP2E1 expression and the activation of ERK, PKC-É and PKC-δ, whereas AMPK knockdown exacerbated the acrylamide-induced neurotoxic effects. PKC-É, but not PKC-δ, plays an upstream regulator of ERK and AMPK. Moreover, AMPK activation suppressed ERK, and CYP2E1 and AMPK bilaterally inhibit each other. Furthermore, acrylamide increased autophagy with impaired autophagic flux, evidenced by the increased beclin-1, LC3-II and p62 protein. Acrylamide-induced neuronal death was ameliorated by 3-methyladenine, an autophagy inhibitor, whereas neuronal death was exacerbated by chloroquine, a lysosomal inhibitor. Interestingly, PKC-δ siRNA, but not PKC-É siRNA, dramatically reduced acrylamide-induced beclin-1 and LC3-II levels, whereas AMPK siRNA further increased beclin-1, LC3-II and p62 protein levels. Glycidamide, a major metabolite, mimicked acrylamide only with a higher potency. Taken together, acrylamide- and glycidamide-induced neurotoxicity may involve cytotoxic autophagy, which is mediated by interplay between PKCs and AMPK pathways.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acrilamida/toxicidade , Compostos de Epóxi/toxicidade , Proteína Quinase C/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Autofagia/efeitos dos fármacos , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Síndromes Neurotóxicas/metabolismo , Células PC12 , Proteína Quinase C/genética , RNA Interferente Pequeno/genética , RatosRESUMO
Mistletoe extracts (Viscum album L.) have been widely used as complementary and alternative medicines for the treatment of cancer, and their cytotoxic effects have been reported on various types of cancer. However, the molecular targets of mistletoe extracts have not been well studied. Herein, we investigated molecules associated with the in vitro and in vivo anticancer effects of mistletoe extract using 4T1 murine breast cancer cells. Mistletoe extract induced apoptosis and inhibited the signal transducer and activator of transcription3 (STAT3) phosphorylation. This inhibition was accompanied by the downregulations of forkhead box M1 (FOXM1) and the DNA repair proteins, RAD51 and survivin. Mistletoe extract simultaneously increased the expression of the DNA damage marker proteins, phosphorylated H2A histone family member X (H2A.X), and phosphorylated p38. Furthermore, mistletoe extract effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition to tumor growth inhibition, mistletoe extract inhibited lung metastasis in the tumor-bearing mice and cell invasiveness by downregulating the expressions of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), uPA receptor, and markers of epithelial-mesenchymal transition (snail and fibronectin). Taken together, our results suggest that mistletoe extract targets the STAT3-FOXM1 pathway for its cytotoxic effects, and that mistletoe extracts might be useful for the treatment of patients with cancers highly expressing the STAT3-FOXM1 pathway.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proteína Forkhead Box M1/metabolismo , Erva-de-Passarinho , Extratos Vegetais/farmacologia , Fator de Transcrição STAT3/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for Galpha(q), which is involved in regulating various vascular functions. To understand how RGS2 regulates foam cell formation, the present study identified signaling pathways controlled by lipopolysaccharide (LPS) and discovered new mechanisms whereby protein kinase C (PKC)-eta and phospholipase D (PLD) 2 regulate RGS2 expression. The toll-like receptor (TLR) 4 agonist LPS caused foam cell formation of Raw264.7 macrophages and dramatically decreased RGS2 mRNA expression. RGS2 down-regulation by LPS was partially recovered by TLR4 small interfering RNA (siRNA). Peritoneal macrophages were separated from wild-type and TLR4 mutant mice, and treatment with LPS showed RGS2 expression decrease in wild-type macrophages but no change in TLR4 mutant macrophages. RGS2 overexpression was suppressed, whereas RGS2 down-regulation accelerated foam cell formation by LPS. Treatment of PKC-eta pseudosubstrate weakened foam cell formation and recovered RGS2 down-regulation by LPS. In addition, LPS or phorbol 12-myristate 13-acetate stimulated PLD activity, and the pretreatment of PLD inhibitor weakened foam cell formation and recovered RGS2 down-regulation. Inhibition of PLD2 expression by siRNA also weakened foam cell formation and partially recovered LPS-mediated RGS2 down-regulation. On the other hand, PLD2 overexpression intensified RGS2 down-regulation and foam cell formation by LPS. These results suggest that LPS causes foam cell formation by increasing PKC-eta and PLD2 activity by down-regulating RGS2 expression via TLR4 dependently.
Assuntos
Células Espumosas/imunologia , Transdução de Sinais/imunologia , Animais , Regulação para Baixo/efeitos dos fármacos , Células Espumosas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/imunologia , Proteínas Ativadoras de GTPase/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Fosfolipase D/genética , Fosfolipase D/imunologia , Fosfolipase D/metabolismo , Proteína Quinase C , Proteínas RGS , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Acetato de Tetradecanoilforbol/imunologia , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
Axl, a member of the TAM (Tyro3, AXL, Mer) receptor tyrosine kinase family, plays critical roles in cell growth, proliferation, apoptosis, and migration. In the present study, we demonstrated that the anti-cancer activity of bufalin, a major bioactive component of the Chinese traditional medicine Chan Su, is mediated by the down-regulation of Axl in non-small-cell lung cancer (NSCLC) cells. We observed the inhibitory effect of bufalin on the proliferation of A549 and H460 NSCLC cells and the clonogenicity of these cells was reduced by bufalin treatment in a dose-dependent manner. Next, we found that the protein level of Axl was decreased in proportion to the concentration of bufalin in both A549 and H460 cells. Moreover, the promoter activity of the Axl gene was decreased by bufalin in a dose- and time-dependent manner, indicating that bufalin down-regulates Axl gene expression at the transcriptional level. We further examined if the anti-proliferative property of bufalin is influenced by Axl at the protein level. Axl overexpression attenuated the effect of bufalin in inhibiting cell proliferation and colony formation and inducing apoptosis in H460 cells, while knockdown of Axl gene expression induced the opposite effect. Taken together, our data indicate that the anti-proliferative and pro-apoptotic effects of bufalin were associated with the protein level of Axl, suggesting that Axl is a potent therapeutic target of bufalin in suppressing proliferation and inducing apoptosis in NSCLC cells.
Assuntos
Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Bufanolídeos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND/AIM: Although molecular targeting therapy is an attractive treatment for cancer, resistance eventually develops in most cases. Here, we evaluated chemotherapeutic efficacy on non-small cell lung cancer (NSCLC) with acquired resistance to epidermal growth factor receptor inhibitors mechanistically. MATERIALS AND METHODS: Antitumor effects of taxotere were evaluated using multiple models, including xenograft, and patient-derived models developed from adenocarcinoma cancer patients. Protein expressions were analyzed after drug treatment. RESULTS: Taxotere inhibited tumor growth of NSCLC cells harboring drug resistance, and reduced the expression of phosphorylated MET proto-oncogene, receptor tyrosine kinase (MET). A tumor-inhibitory effect of taxotere was also demonstrated in vivo in xenografts in mice, patient-derived primary lung tumor cells and patient-derived xenograft with concomitant repression of phosphorylated MET expression. Chemotherapeutic and MET-targeting drug exhibited a synergistic cell growth-inhibitory effect. CONCLUSION: These results suggest that the anticancer drug taxane may be an adjuvant for lung tumors exhibiting enhanced signaling of MET networks.
Assuntos
Antineoplásicos/farmacologia , Docetaxel/farmacologia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To understand the process and molecular mechanism of cellular aging, we explored novel senescence-associated genes using rat tissues with different age. Using total RNAs from 6 and 24-month old rat tissues, differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) were performed and 12 differentially expressed genes (DEGs) which were down- or up-regulated in aged rat tissues were found. Among these DEGs, the level of prosaposin mRNA was elevated of in senescent human dermal fibroblast (HDF) and human umbilical vein endothelial cells (HUVECs). It was further confirmed that expression of prosaposin was up-regulated in prematurely senescent HUVECs induced by hydrogen peroxide or interferon-gamma treatment. Taken together, we report the up-regulation of prosaposin in the senescent HDF and HUVECs as well as in hydrogen peroxide or interferon-gamma induced prematurely senescent HUVECs, suggesting that prosaposin might be a novel senescence-associated gene.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saposinas/genética , Fatores Etários , Animais , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Interferon gama/metabolismo , Rim/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Reprodutibilidade dos Testes , Saposinas/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFRTKI) is an excellent therapeutic agent to treat EGFR mutationpositive nonsmall cell lung cancer (NSCLC). However, the initial response decreases as chemoresistance develops. In the present study, gefitinibresistant EGFR mutant NSCLC PC9/GR cells were established to examine the characteristics and mechanisms associated with chemoresistance. Axl expression in PC9/GR cells was transcriptionally upregulated, since Axl protein and mRNA expression levels were identified to be increased according to western blot analysis and reverse transcription polymerase chain reaction results. The inhibitory effect of celastrol on Axl protein expression level, cell viability and clonogenicity were identified in parental and gefitinibresistant PC9 cells. In addition, treatment of PC9/GR cells with celastrol and gefitinib in combination was demonstrated to synergistically suppress Axl protein expression level, cell proliferation and migration. Taken together, upregulation of Axl expression seems to be associated with chemoresistance of PC9/GR cells. Furthermore, celastrol targets Axl to exert its anticancer effects in order to increase the susceptibility of PC9/GR cells to gefitinib and overcome chemoresistance.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Mutação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Triterpenos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Triterpenos Pentacíclicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptor Tirosina Quinase AxlRESUMO
Recent studies have reported that metformin (Met), the first-line medication for the treatment of type 2 diabetes, exhibited anticancer and chemoprotective effects in diverse cancer cells. In this study, we investigated the effects of Met on the drug-resistance of 4T1 murine breast cancer tumorspheres (TS) and the mechanism responsible for its drug-resistance. 4T1 TS exhibited accumulations of cells at the G0/G1 phase compared with cells in monolayer culture, which suggested the majority of cells in TS were quiescent. Furthermore, it was identified that activations of the signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) signaling pathways in 4T1 TS conferred drug-resistance to doxorubicin (Dox) and lapatinib (Lapa). However, Met selectively targeted TS rather than cells in monolayer culture and increased the cytotoxic effect of Dox on TS by inhibiting activations of the STAT3 and AKT signaling pathways. These observations suggested that inhibitions of STAT3 and AKT underlie the selective cytotoxic effects of Met on TS. In addition, Met exhibited synergistic antitumor effects with Dox on 4T1 tumor-bearing BALB/c mice. Our findings suggest that combinations of Met and cytotoxic anticancer drugs may offer an advantage for treating drug-resistant breast cancer.
RESUMO
Cellular senescence is regulated by specific genes in many organisms. The identification and functional analysis of senescence-associated genes could provide valuable insights into the senescence process. Here, we employed a new and improved differential display reverse transcription-polymerase chain reaction (DDRT-PCR) method that involves annealing control primers (ACPs) to identify genes that are differentially expressed in human umbilical endothelial cells during replicative senescence. Using 120 ACPs, we identified 31 differentially expressed genes (DEGs). Basic local alignment search tool (BLAST) search revealed 29 known genes and two unknown genes. Expression levels of the 29 known genes were confirmed by real-time quantitative RT-RCR and by Western blotting for eight of these genes. CD9 antigen, MHC class I chain-related sequence A (MICA) and cell division cycle 37 homolog (CDC37) were up-regulated, and bone morphogenetic protein 4 (BMP4), dickkopf-1 (DKK1), and transcription factor 7-like 1 (TCF7L1) were down-regulated in old cells. Treatment with recombinant human MICA caused a decrease in cell proliferation and an increase in senescence-associated beta-galactosidase staining. Further analysis of differentially expressed genes may provide insights into the molecular basis of replicative senescence and vascular diseases associated with cellular senescence.
Assuntos
Senescência Celular/genética , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Veias Umbilicais/metabolismo , Antígenos CD/metabolismo , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Primers do DNA , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Transcrição TCF/metabolismo , Tetraspanina 29 , Proteína 1 Semelhante ao Fator 7 de Transcrição , Regulação para CimaRESUMO
We investigated the effect of electromyography (EMG)-triggered neuromuscular electrical stimulation (NMES; EMG-stim) on functional recovery of the hemiparetic hand and the related cortical activation pattern in chronic stroke patients. We enrolled 14 stroke patients, who were randomly assigned to the EMG-stim (n=7) or the control groups (n=7). The EMG-stim was applied to the wrist extensor of the EMG-stim group for two sessions (30 min/session) a day, five times per week for 10 weeks. Four functional tests (box and block, strength, the accuracy index, and the on/offset time of muscle contraction) and functional MRI (fMRI) were performed before and after treatment. fMRI was measured at 1.5 T in parallel with timed finger flexion-extension movements at a fixed rate. Following treatment, the EMG-stim group showed a significant improvement in all functional tests. The main cortical activation change with such functional improvement was shifted from the ipsilateral sensorimotor cortex (SMC) to the contralateral SMC. We demonstrated that 10-week EMG-stim can induce functional recovery and change of cortical activation pattern in the hemiparetic hand of chronic stroke patients.
Assuntos
Encéfalo/fisiopatologia , Terapia por Estimulação Elétrica , Recuperação de Função Fisiológica/fisiologia , Reabilitação do Acidente Vascular Cerebral , Eletromiografia , Feminino , Lateralidade Funcional , Mãos/fisiologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Transcranial direct current stimulation (tDCS) can modulate motor cortex excitability in the human brain. We attempted to demonstrate the cortical stimulation effect of tDCS on the primary motor cortex (M1) using functional MRI (fMRI). An fMRI study was performed for 11 right-handed healthy subjects at 1.5 T. Anodal tDCS was applied to the scalp over the central knob of the M1 in the left hemisphere. A constant current with an intensity of 1.0 mA was applied. The total fMRI paradigm consisted of three sessions with a 5-min resting period between each session. Each session consisted of five successive phases (resting-tDCS-tDCS-tDCS-tDCS), and each of the phases was performed for 21s. Our findings revealed that no cortical activation was detected in any of the stimulation phases except the fourth tDCS phase. In the result of group analysis for the fourth tDCS phase, the average map indicated that the central knob of the left primary motor cortex was activated. In addition, there were activations on the left supplementary motor cortex and the right posterior parietal cortex. We demonstrated that tDCS has a direct stimulation effect on the underlying cortex. It seems that tDCS is a useful modality for stimulating a target cortical region.
Assuntos
Mapeamento Encefálico/métodos , Terapia por Estimulação Elétrica/métodos , Imageamento por Ressonância Magnética/métodos , Córtex Motor/fisiologia , Movimento/fisiologia , Adulto , Circulação Cerebrovascular/fisiologia , Eletricidade , Eletrônica Médica/instrumentação , Eletrônica Médica/métodos , Potencial Evocado Motor/fisiologia , Feminino , Lateralidade Funcional/fisiologia , Humanos , Masculino , Córtex Motor/anatomia & histologia , Córtex Motor/irrigação sanguínea , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Tratos Piramidais/fisiologia , Resultado do TratamentoRESUMO
OBJECTIVES: Constraint-induced movement therapy (CIMT) has been demonstrated to be effective in improving hemiparetic upper extremity function in stroke patients, but few studies have been performed to assess orthosis modification. We investigated the effect of the newly designed small orthosis named modified opposition restriction orthosis (MORO) in chronic hemiparetic patients with stroke. DESIGN: Twenty-one stroke patients were randomly assigned to the CIMT group or control group. Thirteen patients in the CIMT group wore MORO confining the thumb and index finger for at least 5 hours of each day, 7 days a week for 8 weeks. The affected upper extremity function was evaluated using the manual function test (MFT), Purdue Pegboard (PP) score, and motor activity log (MAL) at pre and post-CIMT. RESULTS: Four of the 13 patients in the CIMT group dropped out due to motivational problems, and 9 patients remained in the CIMT group at the end of the study. The patients in the CIMT group showed a mean improvement of 195.8% on MAL AOU (Amount of Use), 24.6% on PP score, and 5.5% on MFT. CONCLUSION: This new MORO would be effective for use in a CIMT program in chronic hemiparetic patients with stroke.
Assuntos
Braquetes , Terapia por Exercício/instrumentação , Hemiplegia/reabilitação , Destreza Motora/fisiologia , Reabilitação do Acidente Vascular Cerebral , Adulto , Idoso , Braço/fisiopatologia , Feminino , Dedos/fisiopatologia , Seguimentos , Hemiplegia/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Atividade Motora/fisiologia , Músculo Esquelético/fisiopatologia , Amplitude de Movimento Articular/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Polegar/fisiopatologiaRESUMO
The aim of this study was to compare the areas of brain activation between complex and simple exercises in a unimanual hand and to assess the possibility of an exercise task for paretic hands following stroke. The subjects included 11 healthy right-handed volunteers. The complex exercise was a wooden ball rotation task with the unimanual hand and the simple exercise was a hand grasp task performed during a functional MRI scan. Stronger activation of the left primary sensorimotor cortex, the left premotor area, and the ipsilateral cerebellum emerged when the complex movement was performed. Ipsilateral activity was located in the primary sensory cortex and premotor area, and contralateral activity was shown in the left cerebellum. These results suggest that a unimanual ball rotation task may be appropriate for rehabilitation of a movable paretic hand in an early stage of stroke recovery, which should provide motor and sensory input using external stimuli, while the simple motor task may appropriate in a compensatory stage, and should inhibit the ipsilateral activity due to maladaptive plasticity.