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Breast cancer remains a global health challenge, and innovative strategies are required to target estrogen receptor α (ERα), a key player in its development. This study investigates the potential of campesterol, a natural phytosterol, as an ERα inhibitor for breast cancer. Our approach integrates in silico, in vitro, and ex vivo experiments to assess the therapeutic potential of campesterol. In silico analyses highlight campesterol as a promising ERα ligand with favorable binding affinities and dynamic properties. Structural analysis reveals conformational changes in ERα upon campesterol binding. In vitro studies confirm the selective growth inhibition of campesterol against ERα-positive breast cancer cells. This study extends to ER+ breast cancer patient-derived organoids (PDOs), showing the effectiveness of campesterol in ERα-positive breast cancer PDOs. Importantly, it emphasizes the receptor-specific nature of campesterol, providing insights into its context-dependent action. In conclusion, campesterol displays potential as an ERα inhibitor, offering new avenues for ER+ breast cancer treatment.
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Neoplasias da Mama , Receptor alfa de Estrogênio , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Descoberta de Drogas , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Fitosteróis/farmacologia , Fitosteróis/química , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Simulação de Acoplamento Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Colesterol/análogos & derivadosRESUMO
Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.
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Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , MAP Quinase Quinase Quinases/metabolismo , Estrogênios , Receptores Proteína Tirosina Quinases , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.
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Antineoplásicos Imunológicos , Neoplasias da Mama , Ligante CD27 , Resistencia a Medicamentos Antineoplásicos , MAP Quinase Quinase Quinases , Nanopartículas , Trastuzumab , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ligante CD27/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Ceramidas/química , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/análise , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
The incidence and mortality of hepatocellular carcinoma (HCC) are on a rise in the Western countries including US, attributed mostly to late detection. Sorafenib has been the first-line FDA-approved drug for advanced unresectable HCC for almost a decade, but with limited efficacy due to the development of resistance. More recently, several other multi-kinase inhibitors (lenvatinib, cabozantinib, regorafenib), human monoclonal antibody (ramucirumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab) have been approved as systemic therapies. Despite this, the median survival of patients is not significantly increased. Understanding of the molecular mechanism(s) that govern HCC resistance is critically needed to increase efficacy of current drugs and to develop more efficacious ones in the future. Our studies with sorafenib-resistant (soraR) HCC cells using transcription factor RT2 Profiler PCR Arrays revealed an increase in E26 transformation-specific-1 (Ets-1) transcription factor in all soraR cells. HCC TMA studies showed an increase in Ets-1 expression in advanced HCC compared to the normal livers. Overexpression or knocking down Ets-1 modulated sorafenib resistance-related epithelial-mesenchymal transition (EMT), migration, and cell survival. In addition, the soraR cells showed a significant reduction of mitochondrial damage and mitochondrial reactive oxygen species (mROS) generation, which were antagonized by knocking down Ets-1 expression. More in-depth analysis identified GPX-2 as a downstream mediator of Ets-1-induced sorafenib resistance, which was down-regulated by Ets-1 knockdown while other antioxidant pathway genes were not affected. Interestingly, knocking down GPX2 expression significantly increased sorafenib sensitivity in the soraR cells. Our studies indicate the activation of a novel Ets-1-GPX2 signaling axis in soraR cells, targeting which might successfully antagonize resistance and increase efficacy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Proteína Proto-Oncogênica c-ets-1/genética , Espécies Reativas de Oxigênio , Sorafenibe/farmacologia , Fatores de TranscriçãoRESUMO
BACKGROUND: Aloe vera extract and its bioactive compounds possess anti-proliferative properties against cancer cells. However, no detailed molecular mechanism of action studies has been reported. We have now employed a computational approach to scrutinize the molecular mechanism of lead bioactive compounds from Aloe vera that potentially inhibit DNA synthesis. METHODS: Initially, the anti-proliferative activity of Aloe vera extract was examined in human breast cancer cells (in vitro/in vivo). Later on, computational screening of bioactive compounds from Aloe vera targeting DNA was performed by molecular docking and molecular dynamics simulation. RESULTS: In-vitro and in-vivo studies confirm that Aloe vera extract effectively suppresses the growth of breast cancer cells without significant cytotoxicity towards non-cancerous normal immortal cells. Computational screening predicts that growth suppression may be due to the presence of DNA intercalating bioactive compounds (riboflavin, daidzin, aloin, etc.) contained in Aloe vera. MM/PBSA calculation showed that riboflavin has a higher binding affinity at the DNA binding sites compared to standard drug daunorubicin. CONCLUSIONS: These observations support the hypothesis that riboflavin may be exploited as an anti-proliferative DNA intercalating agent to prevent cancer and is worthy of testing for the management of cancer by performing more extensive pre-clinical and if validated clinical trials.
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Aloe , Neoplasias , Aloe/química , DNA , Humanos , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologiaRESUMO
The floodplain wetland habitat in the lower Gangetic plains of West Bengal played a significant role in protecting from environmental degradation like pollution, lowering groundwater table, natural hazards, and others as well as supports for human wellbeing. Thus, it is needed to investigate the health status of wetlands and suggest restoration strategies to protect the livelihood patterns dependent on wetlands. This paper presents the health of the wetland ecosystem by comprising the wetland ecosystem health index (WHI) in 2011 and 2018 at the block level of Malda district, as a part of the lower Gangetic flood plain using the pressure-state-response model (PSR model) and AHP method. A total number of six Landsat satellite images and statistical census data were used to determine the wetland health. Wetlands are classified as very healthy (2.81-3.33), healthy (2.41-2.80), sub-healthy (2.01-2.40), unhealthy (1.61-2.00), and sick (0-1.60) category on the basis of the wetland ecosystem health index score. The results of this study showed that the wetlands located surrounding English Bazar, Manikchak, Ratua-II, and Kaliachak-II blocks have a sub-healthy to very healthy condition in 2011 but changed to unhealthy to sick category in 2018 due to the increase of rapid urbanization, population density, and development activities. These areas have belonged to the sub-healthy to sick category in the year 2011 as well as 2018 due to high wetland pressure. Our observation reveals that the ecosystem service value provided by wetlands decreased by 62.51% and 20.46 in the observed period. Management of WEH should emphasize on large (>100 ha) and medium (51-100 ha) sizes of wetlands in the Diara region of West Bengal. Developing local-level institutions and setting restoration goals are useful strategies to manage wetland resources, and protecting biodiversity should be guided by the Government organization and NGOs.
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Áreas Alagadas , Conservação dos Recursos Naturais , Inundações , Índia , RiosRESUMO
MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.
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Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Treonina/química , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Artemisinin is the frontline fast-acting anti-malarial against P. falciparum. Emergence and spread of resistant parasite in eastern-India poses a threat to national malaria control programs. Therefore, the objective of our study is to evaluate the artesunate-sulfadoxine-pyrimethamine efficacy in Central India. 180 monoclonal P. falciparum-infected patients received standard ASSP therapy during August 2015-January 2017, soon after diagnosis and monitored over next 42-days. Artemisinin-resistance was assessed through in-vivo parasite clearance half-life (PC1/2), ex-vivo ring-stage survivability (RSA), and genome analysis of kelch13 and other candidate gene (pfcrt, pfmdr1, pfatpase 6, pfdhfr and pfdhps). Of 180 P. falciparum positive patients, 9.5% showed increased PC1/2 (> 5.5 h), among them eleven isolates (6.1%) showed reduced sensitivity to RSA. In 4.4% of cases, parasites were not cleared by 72 h and showed prolonged PC1/2(5.6 h) (P < 0.005) along with significantly higher RSA (2.2%) than cured patients (0.4%). None of day-3 positive isolates contained the pfkelch13 mutation implicated in artemisinin resistance. Parasite recrudescence was observed in 5.6% patients, which was associated with triple dhfr-dhps (A16I51R59N108I164-S436G437K540G581T613) combination mutation. Emergence of reduced sensitivity to artesunate-sulfadoxine-pyrimethamine, in central India highlighted the risk toward spread of resistant parasite across different parts of India. Day-3 positive parasite, featuring the phenotype of artemisinin-resistance without pfkelch13 mutation, suggested kelch13-independent artemisinin-resistance.
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Artemisininas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Adolescente , Adulto , Antimaláricos/farmacologia , Artemisininas/metabolismo , Resistência a Medicamentos/genética , Quimioterapia Combinada/métodos , Feminino , Humanos , Índia/epidemiologia , Repetição Kelch/genética , Malária/tratamento farmacológico , Malária/parasitologia , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Fenótipo , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Polimorfismo Genético/genética , Proteínas de Protozoários/genética , Pirimetamina/uso terapêutico , Sulfadoxina/uso terapêutico , Tetra-Hidrofolato Desidrogenase/genética , Resultado do TratamentoRESUMO
The transcription factor Glioma-Associated Oncogene Homolog 1 (GLI1) is activated by sonic hedgehog (SHH) cascade and is an established driver of pancreatic ductal adenocarcinoma (PDAC). However, therapies targeting upstream hedgehog signaling have shown little to no efficacy in clinical trials. Here, we identify Mixed Lineage Kinase 3 (MLK3) as a druggable regulator of oncogenic GLI1. Earlier, we reported that MLK3 phosphorylated a peptidyl-prolyl isomerase PIN1 on the S138 site, and the PIN1-pS138 translocated to the nucleus. In this report, we identify GLI1 as one of the targets of PIN1-pS138 and demonstrate that PIN1-pS138 is upregulated in human PDAC and strongly associates with the upregulation of GLI1 and MLK3 expression. Moreover, we also identified two new phosphorylation sites on GLI1, T394, and S1089, which are directly phosphorylated by MLK3 to promote GLI1 nuclear translocation, transcriptional activity, and cell proliferation. Additionally, pharmacological inhibition of MLK3 by CEP-1347 promoted apoptosis in PDAC cell lines, reduced tumor burden, extended survival, and reduced GLI1 expression in the Pdx1-Cre x LSL-KRASG12D x LSL-TP53R172H (KPC) mouse model of PDAC. These findings collectively suggest that MLK3 is an important regulator of oncogenic GLI1 and that therapies targeting MLK3 warrant consideration in the management of PDAC patients.
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MAP Quinase Quinase Quinases/genética , Peptidilprolil Isomerase de Interação com NIMA/genética , Neoplasias Pancreáticas/genética , Proteína GLI1 em Dedos de Zinco/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias Pancreáticas/patologia , Fosforilação/genética , Transdução de Sinais/genética , Transcrição Gênica/genética , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of ß-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Berberina/farmacologia , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Neoplasias Hepáticas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.
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INTRODUCTION: Simulators are used for training competencies including blood pressure (BP) measurement. Whether such training translates to competency in real life is unclear. METHODS: We randomized 145 first-year undergraduate medical students to train on a BP simulator or arms of colleagues. After training, all students were assessed for accuracy (within 4 mm Hg above or below assessor's simultaneous recording) and methodology of BP recordings by an objectively structured clinical examination on real patients. RESULTS: Overall, 67 (46.2%) and 92 (63.4%) students accurately measured systolic and diastolic BP, respectively. Forty-three (58.1%) of 74 students in the conventional training recorded systolic BP accurately compared with 24 (33.8%) of 71 students in simulator training (P = 0.005). Diastolic BP was accurately recorded by 56 (75.7%) of 74 students in conventional group as against 36 (50.7%) of 71 students trained on simulator (P = 0.002). Median (interquartile range) objectively structured clinical examination score in conventional group was 8 (7-9) compared with 6 (5-7) in simulator group (P < 0.001). CONCLUSIONS: Simulator-based training did not translate to clinical competence in terms of accuracy and methodology of BP recordings in real-life settings. Notwithstanding its advantages, simulator-based BP training needs integration with human element to make it meaningful and relevant to clinical practice.
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Determinação da Pressão Arterial/métodos , Competência Clínica , Educação de Graduação em Medicina/métodos , Treinamento por Simulação/métodos , Estudantes de Medicina , Determinação da Pressão Arterial/normas , Feminino , Humanos , Masculino , Treinamento por Simulação/normasRESUMO
Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.
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Neoplasias da Mama/patologia , MAP Quinase Quinase Quinases/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Feminino , Humanos , MAP Quinase Quinase Quinases/genética , Camundongos SCID , Fosforilação , Serina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/química , Quinases Ativadas por p21/genética , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
Organization and distribution of lipids in cellular membranes play an important role in a diverse range of biological processes, such as membrane trafficking and signaling. Here, we present the combined experimental and simulated results to elucidate the phase behavioral features of ganglioside monosialo 1 (GM1)-containing mixed monolayer of the lipids 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and cholesterol (CHOL). Two monolayers having compositions DMPC-CHOL and GM1-DMPC-CHOL are investigated at air-water and air-solid interfaces using Langmuir-Blodgett experiments and scanning electron microscopy (SEM), respectively, to ascertain the phase behavior change of the monolayers. Surface pressure isotherms and SEM imaging of domain formation indicate that addition of GM1 to the monolayer at low surface pressure causes a fluidization of the system but once the system attains the surface pressure corresponding to its liquid-condensed phase, the monolayer becomes more ordered than the system devoid of GM1 and interacts among each other more cooperatively. Besides, the condensing effect of cholesterol on the DMPC monolayer was also verified by our experiments. Apart from these, the effects induced by GM1 on the phase behavior of the binary mixture of DMPC-CHOL were studied with and without applying liquid-expanded (LE)-liquid-condensed (LC) equilibrium surface pressure using molecular dynamics (MD) simulation. Our molecular dynamics (MD) simulation results give an atomistic-level explanation of our experimental findings and furnish a similar conclusion.
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Colesterol/química , Dimiristoilfosfatidilcolina/química , Gangliosídeo G(M1)/química , Membranas Artificiais , Animais , Difusão , Gangliosídeo G(M1)/isolamento & purificação , Cabras , Microdomínios da Membrana/química , Simulação de Dinâmica Molecular , Transição de Fase , PressãoRESUMO
Our previous studies indicated that combination of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PPARγ ligand Troglitazone (TZD), can induce significant apoptosis in various TRAIL-resistant prostate and hepatocellular carcinoma (HCC) cells. These also suggested serine/threonine kinase AMP-activated protein kinase (AMPK) to be a mediator of TRAIL-TZD-induced apoptosis. To further validate AMPK's role in TRAIL sensitization, we determined the apoptotic potential of TRAIL in combination with the natural compound Berberine (BBR), the latter being a potent activator of AMPK. These demonstrated a significant reduction of cell viability and induction of apoptosis (increased cleavage of caspase 3, 8, 9) when treated with TRAIL-BBR combination. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN) or following AMPKα knockdown, confirming involvement of AMPK. To identify potential downstream mediators involved, an apoptosis RT2 PCR array analysis was performed. These showed induction of several genes including TNFRSF10B (expresses DR5) and Harakiri following BBR treatment, which were further validated by qPCR analysis. Furthermore, knocking down DR5 expression significantly attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a mediator of this apoptosis. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of ameliorating TRAIL-resistance involving DR5 in advanced cancer.
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Proteínas Quinases Ativadas por AMP/metabolismo , Berberina/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
A synergistic approach was made to develop a highly stable and carbon resistant catalyst system based on cobalt and nickel supported over modified mesoporous silica for the dry reforming of methane (DRM). Modified mesoporous silica is prepared by a hydrothermal method, and the total Co & Ni composition is taken at around 5% by using the deposition-precipitation technique. CO2 reforming with methane was performed at 400-800 °C under atmospheric pressure as well as at a pressure of 1 MPa, keeping the CH4/CO2 ratio equal to unity. The catalyst assembly before and after the reaction was thoroughly characterized by a wide range of analytical techniques including N2 physisorption, XRD, TPR, TPO, TPH, XPS, SEM, TEM, elemental mapping, TG-DTG. The physicochemical characterization results confirmed the homogeneous distribution of nanosized metal particles into the hexagonal framework of modified silica, which plays a vital role towards a stronger metal support interaction that renders carbon deposition upon the active metal surface as well as avoids metal sintering at higher temperatures. At the same time, the coexistence of nanosized Co and Ni into the mesopores produced a synergy which provides better stability without any deactivation at high pressure reaction conditions. In situ DRIFT analysis evidenced that the reaction proceeds over these catalysts through an initial pathway in which both methane and carbon dioxide initially dissociate over the metal along with a bifunctional pathway in which methane dissociates over the active metal and carbon dioxide activated over the basic support surface via a formate intermediate. Density Functional Theory (DFT) calculations were also performed and further support the proposed mechanism from DRIFT studies.
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Livestock waste composts with minimum inorganic fertilizer as a soil amendment in low-input intensive farming are a feasible agricultural practice to improve soil fertility and productivity and to mitigate soil degradation. The key benefits of the practice rely on the activities of soil microorganisms. However, the role of different livestock composts [composted cattle manure (CCM) vs. composted swine manure (CSM)] on soil microbes, their activities and the overall impact on soil fertility and productivity in a flooded paddy remains elusive. This study compares the effectiveness of CCM and CSM amendment on bacterial communities, activities, nutrient availability, and crop yield in a flooded rice cropping system. We used deep 16S amplicon sequencing and soil enzyme activities to decipher bacterial communities and activities, respectively. Both CCM and CSM amendment significantly increased soil pH, nutrient availability (C, N, and P), microbial biomass, soil enzyme activities indicative for C and N cycles, aboveground plant biomass and grain yield. And the increase in above-mentioned parameters was more prominent in the CCM treatment compared to the CSM treatment. The CCM amendment increased species richness and stimulated copiotrophic microbial groups (Alphaproteobacteria, Betaproteobacteria, and Firmicutes) which are often involved in degradation of complex organic compounds. Moreover, some dominant species (e.g., Azospirillum zeae, Azospirillum halopraeferens, Azospirillum rugosum, Clostridium alkalicellulosi, Clostridium caenicola, Clostridium termitidis, Clostridium cellulolyticum, Magnetospirillum magnetotacticum, Pleomorphomonas oryzae, Variovorax boronicumulans, Pseudomonas xanthomarina, Pseudomonas stutzeri, and Bacillus niacini) which have key roles in plant growth promotion and/or lignocellulose degradation were enhanced under CCM treatment compared to CSM treatment. Multivariate analysis revealed that soil pH and available carbon (C) and nitrogen (N) were the major, while total organic carbon (TOC), total nitrogen (TN), and available phosphorus (P) were the minor drivers of variation in bacterial communities. Overall, our observations suggest that CCM amendment is better than CSM amendment to improve soil fertility and crop yield in a submerged rice cropping system.
RESUMO
Mixed Lineage Kinase 3 (MLK3), also called as MAP3K11 is a tightly regulated MAP3K member but its cellular function is still not fully understood. Earlier we reported post-translational regulation of MLK3 by estrogen (E2) that inhibited the kinase activity and favored survival of ER+ breast cancer cells. Here we report that MLK3 is also transcriptionally downregulated by E2 in ER+ breast cancer cells. Publicly available data and in situ hybridization of human breast tumors showed significant down regulation of MLK3 transcripts in ER+ tumors. The basal level of MLK3 transcripts and protein in ER+ breast cancer cell lines were significantly lower, and the protein expression was further down regulated by E2 in a time-dependent manner. Analysis of the promoter of MLK3 revealed two ERE sites which were regulated by E2 in ER+ but not in ER- breast cancer cell lines. Both ERα and ERß were able to bind to MLK3 promoter and recruit nuclear receptor co-repressors (NCoR, SMRT and LCoR), leading to down-regulation of MLK3 transcripts. Collectively these results suggest that recruitment of nuclear receptor co-repressor is a key feature of ligand-dependent transcriptional repression of MLK3 by ERs. Therefore coordinated transcriptional and post-translational repression of pro-apoptotic MLK3 probably is one of the mechanisms by which ER+ breast cancer cells proliferate and survive.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase Quinases/genética , Receptores de Estrogênio/metabolismo , Transcrição Gênica , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase Quinases/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismo , Elementos de Resposta , Ativação Transcricional , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
CONTEXT: Endodontic retreatment is performed in teeth with endodontic failures. The main goal of retreatment is cleaning and shaping of the root canal with removal of old root filling material. Hand instruments and rotary instruments are mainly used for removing this filling material. AIM OF STUDY: To compare the relative efficacy of three rotary instrumentation systems for removal of gutta-percha from root canal during endodontic retreatment. OBJECTIVE OF STUDY: To find out which NiTi system is more efficacious in retreatment and to check out the efficacy of retreatment with and without use of solvent. MATERIALS AND METHODS: Sixty freshly extracted, single-rooted human mandibular premolars were instrumented with K-files, and each root canal was filled with gutta-percha and AH Plus (Dentsply Detrey, Konstanz, Germany) sealer using lateral compaction. Specimens were then divided into three experimental groups with twenty specimens each. Groups were then subdivided into ten specimens each. Groups were then retreated either with or without solvent. The removal of gutta-percha was performed using ProTaper retreatment files, Mtwo retreatment files, and R-Endo files after 2 weeks. The amount of root canal filling material remnant in the coronal, middle, and apical thirds was measured using stereomicroscope and computer image analysis program. STATISTICAL ANALYSIS USED: Data were evaluated statistically using analysis of variance. RESULTS: ProTaper group was found to have less remnant filling material as compared to the other groups in coronal and middle thirds, but a significant difference was observed between ProTaper and Mtwo and Mtwo and R-Endo in the nonsolvent groups (P < 0.05). Mtwo group demonstrated less amount of remaining filling material in the nonsolvent group. CONCLUSIONS: Both nickel-titanium systems and ProTaper and Mtwo retreatment file systems, were found to be effective in the removal of root canal filling material. However, complete removal of gutta-percha from root canals did not occur with any of the experimental groups.