RESUMO
Although venous invasion (VI) is common in colorectal cancers (CRCs) and is associated with distant metastasis, the 3-dimensional (3D) microscopic features and associated mechanisms of VI are not well elucidated. To characterize the patterns of VI, 103 tissue slabs were harvested from surgically resected CRCs with ≥pT2. They were cleared using the modified immunolabeling-enabled 3D imaging of solvent-cleared organs method, labeled with multicolor fluorescent antibodies, including antibodies against cytokeratin 19, desmin, CD31, and E-cadherin, and visualized by confocal laser scanning microscopy. VI was classified as intravasation, intraluminal growth, and/or extravasation, and 2-dimensional and 3D microscopic features were compared. VI was detected more frequently in 3D (56/103 [54.4%]) than in conventional 2-dimensional hematoxylin and eosin-stained slides (33/103 [32%]; P < .001). When VI was present, it was most commonly in the form of intraluminal growth (51/56), followed by extravasation (13/56) and intravasation (5/56). The mean length of intraluminal growth was 334.0 ± 212.4 µm. Neoplastic cell projections extended from cancer cell clusters in the connective tissue surrounding veins, penetrated the smooth muscle layer, and then grew into and filled the venous lumen. E-cadherin expression changed at each invasion phase; intact E-cadherin expression was observed in the cancer cells in the venous walls, but its expression was lost in small clusters of intraluminal neoplastic cells. In addition, reexpression of E-cadherin was observed when cancer cells formed well-oriented tubular structures and accumulated and grew along the luminal side of the venous wall. In contrast, singly scattered cancer cells and cancer cells with poorly defined tubular structures showed loss of E-cadherin expression. E-cadherin expression was intact in the large cohesive clusters of extravasated cancer cells. However, singly scattered cells and smaller projections of neoplastic cells in the stroma outward of venous wall showed a loss of E-cadherin expression. In conclusion, VI was observed in more than half of the CRCs analyzed by 3D histopathologic image reconstruction. Once inside a vein, neoplastic cells can grow intraluminally. The epithelial-mesenchymal transition is not maintained during VI of CRCs.
Assuntos
Caderinas , Neoplasias Colorretais , Humanos , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Linhagem Celular Tumoral , Neoplasias Colorretais/cirurgia , Neoplasias Colorretais/patologiaRESUMO
The transcription factor FOXG1 plays an important role in inner ear development; however, the cis-regulatory mechanisms controlling the inner-ear-specific expression of FOXG1 are poorly understood. In this study, we aimed to identify the element that specifically regulates FoxG1 expression in the otic vesicle, which develops into the inner ear, through comparative genome analysis between vertebrate species and chromatin immunoprecipitation. The cis-regulatory element (E2) identified showed high evolutionary conservation among vertebrates in the genomic DNA of FoxG1 spanning approximately 3 Mbp. We identified core sequences important for the activity of the otic-vesicle-specific enhancer through in vitro and in vivo reporter assays for various E2 enhancer mutants and determined the consensus sequence for SOX DNA binding. In addition, SoxE, a subfamily of the Sox family, was simultaneously expressed in the otic vesicles of developing embryos and showed a similar protein expression pattern as that of FoxG1. Furthermore, SOXE transcription factors induced specific transcriptional activity through the FoxG1 Otic enhancer (E2b). These findings suggest that the interaction between the otic enhancer of FoxG1 and SOXE transcription factor, in which the otic expression of FoxG1 is evolutionarily well-conserved, is important during early development of the inner ear, a sensory organ important for survival in nature.
Assuntos
Orelha Interna , Fatores de Transcrição SOXE , Animais , DNA/metabolismo , Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/metabolismoRESUMO
The androgen receptor (AR) plays an important role in the pathogenesis and development of prostate cancer (PCa). Mostly, PCa progresses to androgen-independent PCa, which has activated AR signaling from androgen-dependent PCa. Thus, inhibition of AR signaling may be an important therapeutic target in androgen-dependent and castration-resistant PCa. In this study, we determined the anticancer effect of a newly found natural compound, sakurasosaponin (S-saponin), using androgen-dependent and castration-resistant PCa cell lines. S-saponin induces mitochondrial-mediated cell death in both androgen-dependent (LNCaP) and castration-resistant (22Rv1 and C4-2) PCa cells, via AR expression. S-saponin treatment induces a decrease in AR expression in a time- and dose-dependent manner and a potent decrease in the expression of its target genes, including prostate-specific antigen (PSA), transmembrane protease, serin 2 (TMPRSS2), and NK3 homeobox 1 (NKX3.1). Furthermore, S-saponin treatment decreases B-cell lymphoma-extra large (Bcl-xL) and mitochondrial membrane potential, thereby increasing the release of cytochrome c into the cytosol. Moreover, Bcl-xL inhibition and subsequent mitochondria-mediated cell death caused by S-saponin were reversed by Bcl-xL or AR overexpression. Interestingly, S-saponin-mediated cell death was significantly reduced by a reactive oxygen species (ROS) scavenger, N-acetylcystein. Animal xenograft experiments showed that S-saponin treatment significantly reduced tumor growth of AR-positive 22Rv1 xenografts but not AR-negative PC-3 xenografts. Taken together, for the first time, our results revealed that S-saponin induces mitochondrial-mediated cell death in androgen-dependent and castration-resistant cells through regulation of AR mechanisms, including downregulation of Bcl-xL expression and induction of ROS stress by decreasing mitochondrial membrane potential.
Assuntos
Antineoplásicos/intoxicação , Morte Celular/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Saponinas/farmacologia , Androgênios/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Nus , Células PC-3 , Próstata/efeitos dos fármacos , Próstata/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína bcl-X/metabolismoRESUMO
The laminar structure, a unique feature of the mammalian cerebrum, is formed by a number of genes in a highly complex process. The pyramidal neurons that make up each layer of the cerebrum are functionally characterized by specific gene expressions. In particular, Cux1 and Cux2, which are specifically expressed in layer II-IV neurons, are known to regulate dendritic branching, spine morphology, and synapse formation. However, it is still unknown how their expression is regulated transcriptionally. Here we constructed Cux2-mCherry transgenic mice that reproduce the cortical layer II-IV-specific expression of Cux2, a member of the Cut/Cux/CDP family, using BAC transgenesis and a variety of coordinated cortical layer markers that are known to date. Our immunohistochemistry analysis shows that mCherry was expressed in cortical layer II-IV and the corpus callosum in the same way as endogenous Cux2 without ectopic expression. We also identified a region of 220 bp that is highly conserved in mammals and controls specific cerebral expression of Cux2, using comparative genome analysis and in vivo reporter assays. Furthermore, we confirm that Lhx2, whose expression in cortical layer II-IV is similar to that of the Cux2 enhancer, can act as a transcriptional activator. These results suggest that cortical layer II-IV expression of Cux2 can be regulated by the interaction of Cux2-E1 and Lhx2, and that their failure to co-regulate is associated with neurodevelopmental disorders such as autism and schizophrenia.
Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Córtex Cerebral/fisiologia , Cromossomos Artificiais Bacterianos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Luminescentes/genética , Camundongos Transgênicos , Células Piramidais/metabolismo , Fatores de Transcrição/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND/AIMS: Breast cancer is a clinically and molecularly heterogeneous disease. Patients with triple-negative breast cancer (TNBC) have poorer outcomes than those with other breast cancer subtypes due to lack of effective molecular targets for therapy. The present study aimed to the identification of estrogen receptor (ER)ß as a novel mitochondrial target in TNBC cells, together with underlying mechanisms. METHODS: Expression of ERß in clinical breast samples were examined by qRT-PCR, immunohistochemistry and immunoblotting. Subcellular distribution and binding of ERß-Grp75 was determined by confocal microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. The effect of mitocondrial ERß(mitoERß) overexpression on cell proliferation and cell cycle distribution were assessed CCK-8 assays and FACS. Mitochondrial ROS, membrane potential, and Ca²âº level were measured using the specific fluorescent probes Mito-Sox, TMRE, and Rhod-2AM. The tumorigenic effect of mitoERß overexpression was assessed using an anchorage-independent growth assay, sphere formation and a mouse orthotopic xenograft model. RESULTS: ERß expression was lower in tumor tissue than in adjacent normal tissue of patients with breast cancer, and low levels of mitochondrial ERß (mitoERß) also were associated with increased tumor recurrence after surgery. Overexpression of mitoERß inhibited the proliferation of TNBC cells and tumor masses in an animal model. Moreover, overexpression of mitoERß increased ATP production in TNBC cells and normal breast MCF10A cells, with the latter completely reversed by mitoERß knockdown in MCF10A cells. Grp75 was found to positively regulate ERß translocation into mitochondria via a direct interaction. Coimmunoprecipitation and subcellular fractionation experiments revealed that ERß-Grp75 complex is stable in mitochondria. CONCLUSION: These results suggest that the up-regulation of mitoERß in TNBC cells ensures proper mitochondrial transcription, activating the OXPHOS system to produce ATP. Studying the effects of mitoERß on mitochondrial activity and specific mitochondrial gene expression in breast cancer might help predict tumor recurrence, inform clinical decision-making, and identify novel drug targets in the treatment of TNBC.
Assuntos
Trifosfato de Adenosina/biossíntese , Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Cálcio/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Feminino , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Estadiamento de Neoplasias , Fosforilação Oxidativa , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.
Assuntos
Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Neurônios/metabolismo , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Neurônios/química , Neurônios/citologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Epithelial to mesenchymal transition (EMT) is a critical step in the metastasis of epithelial cancer cells. Butyrate, which is produced from dietary fiber by colonic bacterial fermentation, has been reported to influence EMT. However, some studies have reported that butyrate promotes EMT, while others have reported an inhibitory effect. To clarify these controversial results, it is necessary to elucidate the mechanism by which butyrate can influence EMT. In this study, we examined the potential role of annexin A1 (ANXA1), which was previously reported to promote EMT in breast cancer cells, as a mediator of EMT regulation by butyrate. We found that ANXA1 mRNA and protein were expressed in highly invasive melanoma cell lines (A2058 and A375), but not in SK-MEL-5 cells, which are less invasive. We also showed that butyrate induced ANXA1 mRNA and protein expression and promoted EMT-related cell invasion in SK-MEL-5 cells. Downregulation of ANXA1 expression using specific small interfering RNAs in butyrate-treated SK-MEL-5 cells resulted in increased expression of the epithelial marker E-cadherin and decreased cell invasion. Moreover, overexpressing ANXA1 decreased the expression of the E-cadherin. Collectively, these results indicate that butyrate induces the expression of ANXA1 in human melanoma cells, which then promotes invasion through activating the EMT signaling pathway.
Assuntos
Anexina A1/biossíntese , Butiratos/farmacologia , Caderinas/biossíntese , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/patologia , Anexina A1/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Melanoma/genética , Invasividade Neoplásica/patologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Neoplasias Cutâneas , Regulação para Cima/efeitos dos fármacos , Melanoma Maligno CutâneoRESUMO
Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.
Assuntos
Asparaginase/metabolismo , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Asparaginase/deficiência , Asparaginase/genética , Morte Celular , Proteínas de Ligação a DNA , Granzimas/metabolismo , Hipocampo/enzimologia , Chaperonas de Histonas , Humanos , Concentração de Íons de Hidrogênio , Isquemia/enzimologia , Isquemia/fisiopatologia , Ácido Caínico/farmacologia , Cinética , Camundongos , Camundongos Knockout , Neurônios/patologia , Fármacos Neuroprotetores/metabolismo , Células PC12 , Biossíntese de Proteínas , Ratos , Linfócitos T Citotóxicos/enzimologia , Transcrição GênicaRESUMO
Matrix metalloproteinase-9 (MMP-9) plays a central role in the invasion and metastasis of various types of cancer cells. Here, we demonstrate that glaucine, an alkaloid isolated from the plant Corydalis turtschaninovii tuber (Papaveraceae), can inhibit the migration and invasion of human breast cancer cells. We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner. Results from reporter gene and electrophoretic mobility shift assays revealed that glaucine inhibits MMP-9 expression by suppressing activation of the nuclear transcription factor nuclear factor-κB (NF-κB). Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB. Finally, we also found that glaucine inhibits invasion and MMP-9 expression in the highly metastatic MDA-MB-231 breast cancer cell line. Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness.
Assuntos
Aporfinas/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , NF-kappa B/metabolismo , Aporfinas/química , Neoplasias da Mama/patologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The molecular chaperone heat shock protein 90 (HSP90) is overexpressed in plaques of atherosclerosis patients, and is associated with plaque instability. However, the role of HSP90 in atherosclerosis remains unclear. The present study investigated the effects of HSP90 inhibition on migration and proliferation of vascular smooth muscle cells (VSMCs) and involvement in atherosclerosis. To examine the role of HSP90 in VSMC migration, VSMCs were treated with the specific HSP90 inhibitors, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and STA-9090. Results of a chemotaxis assay showed that the HSP90 inhibitors suppress migration of VSMCs. HSP90 inhibition also prevented invasion and sprout formation of VSMCs via inhibition of matrix metalloproteinase-2 proteolytic activity. Results of a flow cytometric analysis showed that HSP90 inhibition induces cell cycle arrest via regulation of cyclin D3, PCNA and pRb. To investigate the role of HSP90 in the development of atherosclerosis, low-density lipoprotein receptor (LDLR) deficient mice were fed with a high cholesterol diet for 4weeks and treated with 17-AAG for 8weeks. HSP90 inhibition suppressed migration of VSMCs into atherosclerotic plaque lesions in high cholesterol diet-stimulated LDLR(-/-) mice. Inhibition of HSP90 attenuates formation of atherosclerotic plaques via suppression of VSMC migration and proliferation, indicating that HSP90 inhibitors can be used as therapeutic agents for atherosclerosis and in stent restenosis.
Assuntos
Aterosclerose/genética , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/genética , Lactamas Macrocíclicas/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/genética , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/etiologia , Aterosclerose/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colesterol/administração & dosagem , Colesterol/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/patologia , Cultura Primária de Células , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução de Sinais , Triazóis/farmacologiaRESUMO
The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.
Assuntos
Neurofibromina 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinas/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Imuno-Histoquímica , Imunoprecipitação , Mutação , Neurofibromina 2/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Serina/metabolismo , Treonina/metabolismo , WortmaninaRESUMO
Sodium meta-arsenite (NaAsO2), a novel compound synthesized by Komipham International Co. Ltd, is an orally bioavailable, water-soluble trivalent arsenical that has shown potent cytotoxic activity in human solid cancer cells in vitro and in vivo, and is currently undergoing phase I/II clinical trials for the treatment of prostate cancer. In this study, mechanisms of cell death induced by sodium meta-arsenite were investigated. Sodium meta-arsenite reduced cell viability and increased the sub-G1 population in cell cycle analysis in both androgen-sensitive LNCaP and androgen-insensitive CWR22RV1 cells. The apoptosis induced by sodium meta-arsenite was associated with cleavage of caspases 3, 8, and 9, and poly (ADP-ribose) polymerase (PARP) and increased annexin V-positive cells, and was inhibited by the pan-caspase inhibitor Z-VAD-fmk. Sodium meta-arsenite also increased the level of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II and the number of autophagic vacuoles as shown by electron microscopy. Both the autophagy inhibitor 3-methyladenine and the necrosis inhibitor necrostatin-1 blocked cell death induced by sodium meta-arsenite. Moreover, sodium meta-arsenite led to the accumulation of intracellular reactive oxygen species (ROS) and N-acetyl-L-cysteine (NAC), a ROS scavenger, decreased sodium meta-arsenite-induced levels of cleaved PARP and LC3-II. Propidium iodide (PI) staining also showed that NAC restored membrane integrity, damaged by sodium meta-arsenite. Therefore, these results suggest that sodium meta-arsenite induces apoptotic, necrotic, and autophagic cell death through intracellular ROS accumulation in both androgen-sensitive and androgen-insensitive prostate cancer cells and may be used as a new anticancer drug for the treatment of prostate cancer.
Assuntos
Androgênios/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sódio/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Necrose , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
Frequent recurrence and metastasis caused by cancer stem cells (CSCs) are major challenges in lung cancer treatment. Therefore, identifying and characterizing specific CSC targets are crucial for the success of prospective targeted therapies. In this study, it is found that upregulated TOR Signaling Pathway Regulator-Like (TIPRL) in lung CSCs causes sustained activation of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) signaling pathway by binding to CaMKK2, thereby maintaining stemness and survival. CaMKK2-mediated activation of CaM kinase 4 (CaMK4) leads to phosphorylation of cAMP response element-binding protein (CREB) at Ser129 and Ser133, which is necessary for its maximum activation and the downstream constitutive expression of its target genes (Bcl2 and HMG20A). TIPRL depletion sensitizes lung CSCs to afatinib-induced cell death and reduces distal metastasis of lung cancer in vivo. It is determined that CREB activates the transcription of TIPRL in lung CSCs. The positive feedback loop consisting of CREB and TIPRL induces the sustained activation of the CaMKK2-CaMK4-CREB axis as a driving force and upregulates the expression of stemness- and survival-related genes, promoting tumorigenesis in patients with lung cancer. Thus, TIPRL and the CaMKK2 signaling axis may be promising targets for overcoming drug resistance and reducing metastasis in lung cancer.
RESUMO
Protein kinase A (PKA) phosphorylates diverse protein substrates to modulate their function. In this study, we found that PKA specifically phosphorylates the RD1 (repression domain 1) domain of nuclear receptor corepressor (NCoR). We demonstrated that the Serine-70 of NCoR is identified the critical amino acid for PKA-dependent NCoR phosphorylation. Importantly, we found that PKA-dependent phosphorylation enhances the nuclear translocation of NCoR. More importantly, the activation of PKA enhanced the repressive activity of NCoR in a reporter assay and potentiated the antagonist activity in the androgen receptor (AR)-mediated transcription. Taken together, these results uncover a regulatory mechanism by which PKA positively modulates NCoR function in transcriptional regulation in prostate cancer.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Neoplasias da Próstata/enzimologia , Transporte Ativo do Núcleo Celular , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Mutação , Correpressor 1 de Receptor Nuclear/genética , Fosforilação , Regiões Promotoras Genéticas , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/genética , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Serina , Transdução de Sinais , Transcrição Gênica , TransfecçãoRESUMO
The epithelial-mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-ß1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin-Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-ß1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-ß1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-ß1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-ß1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-ß1-induced EMT.
Assuntos
Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Triterpenos/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Cães , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Células Madin Darby de Rim Canino , Invasividade Neoplásica , Triterpenos Pentacíclicos , Fatores de Transcrição da Família SnailRESUMO
Brain-derived neurotrophic factor (BDNF) is a cognate ligand for the TrkB receptor. BDNF and serotonin often function in a cooperative manner to regulate neuronal plasticity, neurogenesis, and neuronal survival. Here we show that NAS (N-acetylserotonin) swiftly activates TrkB in a circadian manner and exhibits antidepressant effect in a TrkB-dependent manner. NAS, a precursor of melatonin, is acetylated from serotonin by AANAT (arylalkylamine N-acetyltransferase). NAS rapidly activates TrkB, but not TrkA or TrkC, in a neurotrophin- and MT3 receptor-independent manner. Administration of NAS activates TrkB in BDNF knockout mice. Furthermore, NAS, but not melatonin, displays a robust antidepressant-like behavioral effect in a TrkB-dependent way. Endogenous TrkB is activated in wild-type C3H/f(+/+) mice but not in AANAT-mutated C57BL/6J mice, in a circadian rhythm; TrkB activation is high at night in the dark and low during the day. Hence, our findings support that NAS is more than a melatonin precursor, and that it can potently activate TrkB receptor.
Assuntos
Antidepressivos/farmacologia , Ritmo Circadiano , Depressão/metabolismo , Melatonina/metabolismo , Receptor trkB/agonistas , Serotonina/análogos & derivados , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Linhagem Celular , Hipocampo/metabolismo , Humanos , Melatonina/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fármacos Neuroprotetores/farmacologia , Receptor trkB/metabolismo , Retina/metabolismo , Serotonina/metabolismo , Serotonina/farmacologiaRESUMO
In the medial prefrontal cortex, the prelimbic area is emerging as a major modulator of fear behavior, but the mechanisms remain unclear. Using a selective neocortical knockout mouse, virally mediated prelimbic cortical-specific gene deletion, and pharmacological rescue with a TrkB agonist, we examined the role of a primary candidate mechanism, BDNF, in conditioned fear. We found consistently robust deficits in consolidation of cued fear but no effects on acquisition, expression of unlearned fear, sensorimotor function, and spatial learning. This deficit in learned fear in the BDNF knockout mice was rescued with systemic administration of a TrkB receptor agonist, 7,8-dihydroxyflavone. These data indicate that prelimbic BDNF is critical for consolidation of learned fear memories, but it is not required for innate fear or extinction of fear. Moreover, use of site-specific, inducible BDNF deletions shows a powerful mechanism that may further our understanding of the pathophysiology of fear-related disorders.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Medo/fisiologia , Aprendizagem/fisiologia , Memória/fisiologia , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/genética , Córtex Cerebral/metabolismo , Condicionamento Psicológico/efeitos dos fármacos , Extinção Psicológica , Medo/efeitos dos fármacos , Medo/psicologia , Feminino , Flavonas/farmacologia , Hibridização In Situ , Aprendizagem/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neocórtex/metabolismo , Receptor trkB/agonistas , Receptor trkB/fisiologiaRESUMO
Brain-derived neurotrophic factor (BDNF), a cognate ligand for the tyrosine kinase receptor B (TrkB) receptor, mediates neuronal survival, differentiation, synaptic plasticity, and neurogenesis. However, BDNF has a poor pharmacokinetic profile that limits its therapeutic potential. Here we report the identification of 7,8-dihydroxyflavone as a bioactive high-affinity TrkB agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signaling. 7,8-Dihydroxyflavone protected wild-type, but not TrkB-deficient, neurons from apoptosis. Administration of 7,8-dihydroxyflavone to mice activated TrkB in the brain, inhibited kainic acid-induced toxicity, decreased infarct volumes in stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson disease. Thus, 7,8-dihydroxyflavone imitates BDNF and acts as a robust TrkB agonist, providing a powerful therapeutic tool for the treatment of various neurological diseases.
Assuntos
Apoptose/efeitos dos fármacos , Flavonas/farmacologia , Neurônios/efeitos dos fármacos , Receptor trkB/agonistas , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Flavonas/química , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND/AIM: Breast cancer is the most common cancer among women and the leading cause of cancer-related deaths worldwide. Despite various therapeutic strategies, its impact on the survival rate and quality of life of patients remains limited. The Forkhead Box J3 (FOXJ3) transcription factor has been implicated in various cancers, including lung cancer, tongue squamous cell carcinoma, prostate cancer, and colorectal cancer. However, the role of FOXJ3 in breast cancer has not been elucidated. This study aimed to investigate the role of FOXJ3 in breast cancer development, migration, and invasion. MATERIALS AND METHODS: FOXJ3 expression was analyzed in patient tissues and breast cancer cell lines. Loss-of-function and gain-of-function studies were performed using MDA-MB-231 and MCF7 cell lines, respectively. Cell proliferation, migration, and invasion assays were conducted, and the effects of FOXJ3 on Snail expression were examined. RESULTS: FOXJ3 is over-expressed in breast cancer tissues compared to normal counterparts and in various breast cancer cell lines. By modulating FOXJ3 expression in breast cancer cell lines, we observed its influence on cell proliferation, migration, and invasion. Microarray analysis and subsequent validation showed that FOXJ3 modulates Snail expression, a well-known transcription factor involved in epithelial-mesenchymal transition. CONCLUSION: FOXJ3 plays a role in cell proliferation, migration, and the regulation of Snail expression and may be a potential therapeutic target for breast cancer treatment.
Assuntos
Neoplasias da Mama , Carcinoma de Células Escamosas , Fatores de Transcrição Forkhead , Fatores de Transcrição da Família Snail , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Qualidade de Vida , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
Sakurasosaponin (S-saponin; PubChem ID: 3085160), a recently identified saponin from the roots of Primula sieboldii, has shown potential anticancer properties against various types of cancer. In the present study, the effects of S-saponin on non-small cell lung cancer (NSCLC) cell proliferation and the underlying mechanisms, were investigated. The effect of S-saponin on cell proliferation and cell death were assessed CCK-8, clonogenic assay, western blotting and Annexin V/PI double staining. S-saponin-induced autophagy was determined by confocal microscopic analysis and immunoblotting. S-saponin inhibited the proliferation of A549 and H1299 NSCLC cell lines in a dose- and time-dependent manner, without inducing apoptosis. S-saponin treatment induced autophagy in these cells, as evidenced by the increased LC3-II levels and GFP-LC3 puncta formation. It activated the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, which is crucial for autophagy induction. Inhibition of AMPK with Compound C or siRNA-mediated knockdown of AMPK abrogated S-saponin-induced autophagy and partially rescued cell proliferation. Therefore, S-saponin exerts anti-proliferative effects on NSCLC cells through autophagy induction via AMPK activation. Understanding the molecular mechanisms underlying the anticancer effects of S-saponin in NSCLC cells could provide insights for the development of novel therapeutic strategies for NSCLC.