RESUMO
Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.
Assuntos
Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Carcinogênese/patologia , Feminino , Deleção de Genes , Humanos , Insulina/metabolismo , Isoenzimas/metabolismo , Metástase Neoplásica , Neutrófilos/metabolismo , Receptor ErbB-2/metabolismoRESUMO
Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placenta, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model. Disruption of AKT1 resulted in placental, fetal and postnatal growth restriction. Forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate, was abundantly expressed in the junctional zone and in invasive trophoblast cells of the rat placentation site. Foxo4 gene disruption using genome editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells, but in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network that reciprocally controls critical indices of hemochorial placenta development.
Assuntos
Placenta , Placentação , Animais , Feminino , Gravidez , Ratos , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Placenta/metabolismo , Placentação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trofoblastos , ÚteroRESUMO
Undifferentiated spermatogonia are composed of a heterogeneous cell population including spermatogonial stem cells (SSCs). Molecular mechanisms underlying the regulation of various spermatogonial cohorts during their self-renewal and differentiation are largely unclear. Here we show that AKT1S1, an AKT substrate and inhibitor of mTORC1, regulates the homeostasis of undifferentiated spermatogonia. Although deletion of Akt1s1 in mouse appears not grossly affecting steady-state spermatogenesis and male mice are fertile, the subset of differentiation-primed OCT4+ spermatogonia decreased significantly, whereas self-renewing GFRα1+ and proliferating PLZF+ spermatogonia were sustained. Both neonatal prospermatogonia and the first wave spermatogenesis were greatly reduced in Akt1s1-/- mice. Further analyses suggest that OCT4+ spermatogonia in Akt1s1-/- mice possess altered PI3K/AKT-mTORC1 signaling, gene expression and carbohydrate metabolism, leading to their functionally compromised developmental potential. Collectively, these results revealed an important role of AKT1S1 in mediating the stage-specific signals that regulate the self-renewal and differentiation of spermatogonia during mouse spermatogenesis.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Espermatogônias , Masculino , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Testículo/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espermatogênese/genética , Diferenciação Celular/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
Potassium (K+), being an essential macronutrient in plants, plays a central role in many aspects. Root growth is highly plastic and is affected by many different abiotic stresses including nutrient deficiency. The Shaker-type K+ channel Arabidopsis (Arabidopsis thaliana) K+ Transporter 1 (AKT1) is responsible for K+ uptake under both low and high external K+ conditions. However, the upstream transcription factor of AKT1 is not clear. Here, we demonstrated that the WRKY6 transcription factor modulates root growth to low potassium (LK) stress in Arabidopsis. WRKY6 showed a quick response to LK stress and also to many other abiotic stress treatments. The two wrky6 T-DNA insertion mutants were highly sensitive to LK treatment, whose primary root lengths were much shorter, less biomass and lower K+ content in roots than those of wild-type plants, while WRKY6-overexpression lines showed opposite phenotypes. A further investigation showed that WRKY6 regulated the expression of the AKT1 gene via directly binding to the W-box elements in its promoter through EMSA and ChIP-qPCR assays. A dual luciferase reporter analysis further demonstrated that WRKY6 enhanced the transcription of AKT1. Genetic analysis further revealed that the overexpression of AKT1 greatly rescued the short root phenotype of the wrky6 mutant under LK stress, suggesting AKT1 is epistatic to WRKY6 in the control of LK response. Further transcriptome profiling suggested that WRKY6 modulates LK response through a complex regulatory network. Thus, this study unveils a transcription factor that modulates root growth under potassium deficiency conditions by affecting the potassium channel gene AKT1 expression.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Raízes de Plantas , Potássio , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Potássio/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Canais de PotássioRESUMO
BACKGROUND: Lymphatic valves are specialized structures in collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation. METHODS: In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium. RESULTS: Here, we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human dermal lymphatic endothelial cells not only reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions but also abolished the upregulation of AKT activity and valve-forming genes in response to oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery. CONCLUSIONS: Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.
Assuntos
Proteínas de Transporte , Proteína Forkhead Box O1 , Linfangiogênese , Vasos Linfáticos , Alvo Mecanístico do Complexo 2 de Rapamicina , Mecanotransdução Celular , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais , Animais , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Vasos Linfáticos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Humanos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Células Endoteliais/metabolismo , Células Cultivadas , Serina-Treonina Quinases TOR/metabolismo , Fosforilação , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/genética , Camundongos Endogâmicos C57BL , Interferência de RNA , TransfecçãoRESUMO
The importance of protein kinase B (AKT) in tumorigenesis and development is well established, but its potential regulation of metabolic reprogramming via phosphorylation of the hexokinase (HK) isozymes remains unclear. There are two HK family members (HK1/2) and three AKT family members (AKT1/2/3), with varied distribution of AKTs exhibiting distinct functions in different tissues and cell types. Although AKT is known to phosphorylate HK2 at threonine 473, AKT-mediated phosphorylation of HK1 has not been reported. We examined direct binding and phosphorylation of HK1/2 by AKT1 and identified the phosphorylation modification sites using coimmunoprecipitation, glutathione pull-down, western blotting, and in vitro kinase assays. Regulation of HK activity through phosphorylation by AKT1 was also examined. Uptake of 2-[1,2-3H]-deoxyglucose and production of lactate were investigated to determine whether AKT1 regulates glucose metabolism by phosphorylating HK1/2. Functional assays, immunohistochemistry, and tumor experiments in mice were performed to investigate whether AKT1-mediated regulation of tumor development is dependent on its kinase activity and/or the involvement of HK1/2. AKT interacted with and phosphorylated HK1 and HK2. Serine phosphorylation significantly increased AKT kinase activity, thereby enhancing glycolysis. Mechanistically, the phosphorylation of HK1 at serine 178 (S178) by AKT significantly decreased the Km and enhanced the Vmax by interfering with the formation of HK1 dimers. Mutations in the AKT phosphorylation sites of HK1 or HK2 significantly abrogated the stimulatory characteristics of AKT on glycolysis, tumorigenesis, and cell migration, invasion, proliferation, and metastasis. HK1-S178 phosphorylation levels were significantly correlated with the occurrence and metastasis of different types of clinical tumors. We conclude that AKT not only regulates tumor glucose metabolism by directly phosphorylating HK1 and HK2, but also plays important roles in tumor progression, proliferation, and migration.
Assuntos
Carcinogênese , Hexoquinase , Proteínas Proto-Oncogênicas c-akt , Hexoquinase/metabolismo , Hexoquinase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Humanos , Animais , Fosforilação , Camundongos , Carcinogênese/metabolismo , Carcinogênese/genética , Metástase Neoplásica , Feminino , Linhagem Celular Tumoral , Proliferação de Células , Movimento Celular , Glucose/metabolismoRESUMO
Sclerosing pneumocytoma is a rare and distinct lung neoplasm whose histogenesis and molecular alterations are the subject of ongoing research. Our recent study revealed that AKT1 internal tandem duplications (ITD), point mutations, and short indels were present in almost all tested sclerosing pneumocytomas, suggesting that AKT1 mutations are a major driving oncogenic event in this tumor. Although the pathogenic role of AKT1 point mutations is well established, the significance of AKT1 ITD in oncogenesis remains largely unexplored. We conducted comprehensive genomic and transcriptomic analyses of sclerosing pneumocytoma to address this knowledge gap. RNA-sequencing data from 23 tumors and whole-exome sequencing data from 44 tumors were used to obtain insights into their genetic and transcriptomic profiles. Our analysis revealed a high degree of genetic and transcriptomic similarity between tumors carrying AKT1 ITD and those with AKT1 point mutations. Mutational signature analysis revealed COSMIC signatures 1 and 5 as the prevailing signatures of sclerosing pneumocytoma, associated with the spontaneous deamination of 5-methylcytosine and an unknown etiology, respectively. RNA-sequencing data analysis revealed that the sclerosing pneumocytoma gene expression profile is characterized by activation of the PI3K/AKT/mTOR pathway, which exhibits significant similarity between tumors harboring AKT1 ITD and those with AKT1 point mutations. Notably, an upregulation of SOX9, a transcription factor known for its involvement in fetal lung development, was observed in sclerosing pneumocytoma. Specifically, SOX9 expression was prominent in the round cell component, whereas it was relatively lower in the surface cell component of the tumor. To the best of our knowledge, this is the first comprehensive investigation of the genomic and transcriptomic characteristics of sclerosing pneumocytoma. Results of the present study provide insights into the molecular attributes of sclerosing pneumocytoma and a basis for future studies of this enigmatic tumor.
Assuntos
Fosfatidilinositol 3-Quinases , Hemangioma Esclerosante Pulmonar , Humanos , Fosfatidilinositol 3-Quinases/genética , Hemangioma Esclerosante Pulmonar/genética , Hemangioma Esclerosante Pulmonar/patologia , Genômica , Perfilação da Expressão Gênica , RNARESUMO
Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3ß levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.
RESUMO
Increasing evidence suggests the oncogenic role of missense mutation (AKT1-E17K) of AKT1 gene in meningiomas. Upon investigating the connection between the pro-tumorigenic role of AKT1-E17K and cellular metabolic adaptations, elevated levels of glycolytic enzyme hexokinase 2 (HK2) was observed in meningioma patients with AKT1-E17K compared to patients harboring wild-type AKT1. In vitro experiments also suggested higher HK2 levels and its activity in AKT1-E17K cells. Treatment with the conventional drug of choice AZD5363 (a pan AKT inhibitor) enhanced cell death and diminished HK2 levels in AKT1 mutants. Given the role of AKT phosphorylation in eliciting inflammatory responses, we observed increased levels of inflammatory mediators (IL-1ß, IL6, IL8, and TLR4) in AKT1-E17K cells compared to AKT1-WT cells. Treatment with AKT or HK2 inhibitors dampened the heightened levels of inflammatory markers in AKT1-E17K cells. As AKT and HK2 regulates redox homeostasis, diminished ROS generation concomitant with increased levels of NF-E2- related factor 2 (Nrf2) and superoxide dismutase 1 (SOD1) were observed in AKT1-E17K cells. Increased sensitivity of AKT1-E17K cells to AZD5363 in the presence of HK2 inhibitor Lonidamine was reversed upon treatment with ROS inhibitor NAC. By affecting metabolism, inflammation, and redox homeostasis AKT1-E17K confers a survival advantage in meningioma cells. Our findings suggest that targeting AKT-HK2 cross-talk to induce ROS-dependent cell death could be exploited as novel therapeutic approach in meningiomas.
Assuntos
Neoplasias Meníngeas , Meningioma , Humanos , Mutação com Ganho de Função , Hexoquinase/genética , Hexoquinase/metabolismo , Neoplasias Meníngeas/genética , Meningioma/genética , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de OxigênioRESUMO
IMP-3 expression is a poor prognostic factor of melanomas and it promotes melanoma cell migration and invasion by a pathway modulating HMGA2 mRNA expression. We tried to identify other putative targets of IMP-3. We identified putative IMP-3-binding RNAs, including AKT1, MAPK3, RB1 and RELA, by RNA immunoprecipitation coupled with next-generation sequencing. IMP-3 overexpression increased AKT and RELA levels in MeWo cells. siRNAs against AKT1 and RELA inhibited MeWo/Full-length IMP-3 cell migration. IMP-3 knockdown of A2058 cells decreased AKT1 and RELA expression and lowered migration ability. Co-transfection of A2058 cells with AKT1- or RELA-expressing plasmids with IMP-3 siRNA restored the inhibitory effects of IMP-3 knockdown on migration. HMGA2 did not influence AKT1 and RELA expression in melanoma cells. Human melanoma samples with high IMP-3 levels also showed high HMGA2, AKT1 and RELA expression. Our results show that IMP-3 enhances melanoma cell migration through the regulation of the AKT1 and RELA axis.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas c-akt , Proteínas de Ligação a RNA , Fator de Transcrição RelA , Humanos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Statin therapy is a cornerstone in the treatment of systemic vascular diseases. However, statins have failed to translate as therapeutics for pulmonary vascular disease. Early pulmonary vascular disease in the setting of congenital heart disease (CHD) is characterized by endothelial dysfunction, which precedes the more advanced stages of vascular remodeling. These features make CHD an ideal cohort in which to re-evaluate the potential pulmonary vascular benefits of statins, with a focus on endothelial biology. However, it is critical that the full gamut of the pleiotropic effects of statins in the endothelium are uncovered. The purpose of this investigation was to evaluate the therapeutic potential of simvastatin for children with CHD and pulmonary over-circulation, and examine mechanisms of simvastatin action on the endothelium. Our data demonstrate that daily simvastatin treatment preserves endothelial function in our shunt lamb model of pulmonary over-circulation. Further, using pulmonary arterial endothelial cells (PAECs) isolated from Shunt and control lambs, we identified a new mechanism of statin action mediated by increased expression of the endogenous Akt1 inhibitor, C-terminal modifying protein (CTMP). Increases in CTMP were able to decrease the Akt1-mediated mitochondrial redistribution of endothelial nitric oxide synthase (eNOS) which correlated with increased enzymatic coupling, identified by increases in NO generation and decreases in NOS-derived superoxide. Together our data identify a new mechanism by which simvastatin enhances NO signaling in the pulmonary endothelium and identify CTMP as a potential therapeutic target to prevent the endothelial dysfunction that occurs in children born with CHD resulting in pulmonary over-circulation.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Doenças Vasculares , Humanos , Criança , Animais , Ovinos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Sinvastatina/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Endotélio/metabolismo , Doenças Vasculares/metabolismo , Óxido Nítrico/metabolismo , Endotélio Vascular/metabolismoRESUMO
Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.
Assuntos
Mastite Bovina , Infecções Estafilocócicas , Feminino , Animais , Bovinos , Humanos , RNA Guia de Sistemas CRISPR-Cas , Proteínas do Leite/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Mastite Bovina/genética , Mastite Bovina/metabolismo , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/metabolismo , Desmetilação , Glândulas Mamárias Animais/metabolismo , Células Epiteliais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Antascomicin B is a natural product that similarly to the macrolides FK506 and Rapamycin binds to the FK506-binding protein 12 (FKBP12). FK506 and Rapamycin act as molecular glues by inducing ternary complexes between FKBPs and additional target proteins. Whether Antascomicin B can induce ternary complexes is unknown. Here we show that Antascomicin B binds tightly to larger human FKBP homologs. The cocrystal structure of FKBP51 in complex with Antascomicin B revealed that large parts of Antascomicin B are solvent-exposed and available to engage additional proteins. Cellular studies demonstrated that Antascomicin B enhances the interaction between human FKBP51 and human Akt. Our studies show that molecules with molecular glue-like properties are more prominent in nature than previously thought. We predict the existence of additional 'orphan' molecular glues that evolved to induce ternary protein complexes but where the relevant ternary complex partners are unknown.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Proteínas de Ligação a Tacrolimo , Tacrolimo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirolimo/farmacologia , Tacrolimo/farmacologia , Tacrolimo/análogos & derivados , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Maladaptive repair after acute kidney injury (AKI) can predispose patients to chronic kidney disease (CKD). However, the molecular mechanism underlying the AKI-to-CKD transition remains unclear. The Akt signaling pathway has been reported to be involved in the pathological processes of both AKI and CKD. In this study, we investigated the role of Akt1 in a murine model of the AKI-to-CKD transition. Wild-type (WT) and Akt1-/- mice were subjected to unilateral ischemia-reperfusion injury (UIRI), with their kidneys harvested after two days and two, four, and six weeks after UIRI. The dynamic changes in tubulointerstitial fibrosis, markers of tubular epithelial-mesenchymal transition (EMT), and tubular apoptosis were investigated. Akt1 of the three Akt isoforms was activated during the AKI-to-CKD transition. After UIRI, tubulointerstitial fibrosis and tubular EMT were significantly increased in WT mice, but were attenuated in Akt1-/- mice. The expression of the transforming growth factor (TGF)-ß1/Smad was increased in both WT and Akt1-/- mice, but was not different between the two groups. The levels of phosphorylated glycogen synthase kinase (GSK)-3ß, Snail, and ß-catenin in the Akt1-/- mice were lower than those in the WT mice. The number of apoptotic tubular cells and the expression of cleaved caspase-3/Bax were both lower in Akt1-/- mice than in WT mice. Genetic deletion of Akt1 was associated with attenuation of tubulointerstitial fibrosis, tubular EMT, and tubular apoptosis during the AKI-to-CKD transition. These findings were associated with TGF-ß1/Akt1/GSK-3ß/(Snail and ß-catenin) signaling independent of TGF-ß1/Smad signaling. Thus, Akt1 signaling could serve as a potential therapeutic target for inhibiting the AKI-to-CKD transition.
Assuntos
Injúria Renal Aguda , Insuficiência Renal Crônica , Camundongos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Modelos Animais de Doenças , Insuficiência Renal Crônica/metabolismo , Rim/metabolismo , Injúria Renal Aguda/metabolismo , Fibrose , Apoptose , Transição Epitelial-MesenquimalRESUMO
Novel thieno[2,3-d]pyrimidine analogues were designed, synthesized and evaluated for anti-proliferative activity against HepG-2, PC-3 and MCF-7 cancer cell lines. In addition, WI-38 normal cell line was used to explore the safety of all the tested compounds. Compounds 2 (IC50 = 4.29 µM HePG-2, 10.84 µM MCF-7), 6 (IC50 = 14.86 µM HePG-2, 8.04 µM PC-3 and 12.90 µM MCF-7) and 17 (IC50 = 9.98 µM HePG-2, 33.66 µM PC-3 and 14.62 µM MCF-7) were the most promising candidates on the tested cancer cells with high selective toxicity-sparing normal cells. A further mechanistic evaluation revealed promising kinase inhibitory activity, where compound 2 inhibited VEGFR-2 and AKT at IC50 = 0.161 and 1.06 µM, respectively, Furthermore, derivative 6 inhibited VEGFR-2 and AKT at IC50 = 0.487 and 0.364 µM, respectively, while compound 17 showed IC50 = 0.164 and 0.452 µM, respectively. Moreover, compounds 2, 6 resulted in G1 phase cell cycle arrest while candidate 17 arrest cell cycle at G2/M phase. Similar to the apoptosis results, compound 17 showed the highest autophagic induction among the evaluated derivatives. Finally, docking studies were conducted to assess the binding patterns of these active derivatives. The results showed that the binding patterns inside the active sites of both the VEGFR-2 and AKT-1 (allosteric pocket) crystal structures were identical to the reference ligands.
Assuntos
Antineoplásicos , Apoptose , Autofagia , Proliferação de Células , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt , Pirimidinas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Humanos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Pirimidinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Apoptose/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Autofagia/efeitos dos fármacos , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Simulação de Acoplamento Molecular , Linhagem Celular TumoralRESUMO
The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.
Assuntos
Antineoplásicos , Proliferação de Células , Neoplasias Colorretais , Depsipeptídeos , Compostos Macrocíclicos , Proteínas Proto-Oncogênicas c-akt , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Depsipeptídeos/farmacologia , Depsipeptídeos/uso terapêutico , Depsipeptídeos/química , Depsipeptídeos/síntese química , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Akt1, as an important member of the Akt family, plays a controlled role in cancer cell growth and survival. Inhibition of Akt1 activity can promote cancer cell apoptosis and inhibit tumor growth. Therefore, in this investigation, a multilayer virtual screening approach, including receptor-ligand interaction-based pharmacophore, 3D-QSAR, molecular docking, and deep learning methods, was utilized to construct a virtual screening platform for Akt1 inhibitors. 17 representative compounds with different scaffolds were identified as potential Akt1 inhibitors from three databases. Among these 17 compounds, the Hit9 exhibited the best inhibitory activity against Akt1 with inhibition rate of 33.08% at concentration of 1 µM. The molecular dynamics simulations revealed that Hit9 and Akt1 could form a compact and stable complex. Moreover, Hit9 interacted with some key residues by hydrophobic, electrostatic, and hydrogen bonding interactions and induced substantial conformation changes in the hinge region of the Akt1 active site. The average binding free energies for the Akt1-CQU, Akt1-Ipatasertib, and Akt1-Hit9 systems were - 34.44, - 63.37, and - 39.14 kJ mol-1, respectively. In summary, the results obtained in this investigation suggested that Hit9 with novel scaffold may be a promising lead compound for developing new Akt1 inhibitor for treatment of various cancers with Akt1 overexpressed.
RESUMO
Akt1 (protein kinase B) has become a major focus of attention due to its significant functionality in a variety of cellular processes and the inhibition of Akt1 could lead to a decrease in tumour growth effectively in cancer cells. In the present work, we discovered a set of novel Akt1 inhibitors by using multiple computational techniques, i.e. pharmacophore-based virtual screening, molecular docking, binding free energy calculations, and ADME properties. A five-point pharmacophore hypothesis was implemented and validated with AADRR38. The obtained R2 and Q2 values are in the acceptable region with the values of 0.90 and 0.64, respectively. The generated pharmacophore model was employed for virtual screening to find out the potential Akt1 inhibitors. Further, the selected hits were subjected to molecular docking, binding free energy analysis, and refined using ADME properties. Also, we designed a series of 6-methoxybenzo[b]oxazole analogues by comprising the structural characteristics of the hits acquired from the database. Molecules D1-D10 were found to have strong binding interactions and higher binding free energy values. In addition, Molecular dynamic simulation was performed to understand the conformational changes of protein-ligand complex.
RESUMO
Rheumatoid Arthritis (RA) is a chronic, symmetrical inflammatory autoimmune disorder characterized by painful, swollen synovitis and joint erosions, which can cause damage to bone and cartilage and be associated with progressive disability. Despite expanded treatment options, some patients still experience inadequate response or intolerable adverse effects. Consequently, the treatment options for RA remain quite limited. The enzyme AKT1 is crucial in designing drugs for various human diseases, supporting cellular functions like proliferation, survival, metabolism, and angiogenesis in both normal and malignant cells. Therefore, AKT serine/threonine kinase 1 is considered crucial for targeting therapeutic strategies aimed at mitigating RA mechanisms. In this context, directing efforts toward AKT1 represents an innovative approach to developing new anti-arthritis medications. The primary objective of this research is to prioritize AKT1 inhibitors using computational techniques such as molecular modeling and dynamics simulation (MDS) and shape-based virtual screening (SBVS). A combined SBVS approach was employed to predict potent inhibitors against AKT1 by screening a pool of compounds sourced from the ChemDiv and IMPPAT databases. From the SBVS results, only the top three compounds, ChemDiv_7266, ChemDiv_2796, and ChemDiv_9468, were subjected to stability analysis based on their high binding affinity and favorable ADME/Tox properties. The SBVS findings have revealed that critical residues, including Glu17, Gly37, Glu85, and Arg273, significantly contribute to the successful binding of the highest-ranked lead compounds at the active site of AKT1. This insight helps to understand the specific binding mechanism of these leads in inhibiting RA, facilitating the rational design of more effective therapeutic agents.
RESUMO
The deadliest type of skin cancer, malignant melanoma, is also the reason for the majority of skin cancer-related deaths. The objective of this article was to investigate the efficiency of free caffeic acid phenethyl ester (CAPE) and liposomal CAPE in inducing apoptosis in melanoma cells (A375) in in vitro. CAPE was loaded into liposomes made up of hydrogenated soybean phosphatidylcholine, cholesterol, and 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], and their physicochemical properties were assessed. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed for comparing the cytotoxicity of free CAPE and liposomal CAPE at dosages of 10, 15, 25, 50, 75 and the highest dose of 100 µg/mL for period of 24 and 48 h on A375 cell line to calculate IC50. Apoptosis and necrosis were evaluated in A375 melanoma cancer cells using flow cytometry. Atomic force microscopy was utilized to determine the nanomechanical attributes of the membrane structure of A375 cells. To determine whether there were any effects on apoptosis, the expression of PI3K/AKT1 and BAX/BCL2 genes was analyzed using the real-time polymerase chain reaction technique. According to our results, the maximum amount of drug release from nanoliposomes was determined to be 91% and the encapsulation efficiency of CAPE in liposomes was 85.24%. Also, the release of free CAPE was assessed to be 97%. Compared with liposomal CAPE, free CAPE showed a greater effect on reducing the cancer cell survival after 24 and 48 h. Therefore, IC50 values of A375 cells treated with free and liposomal CAPE were calculated as 47.34 and 63.39 µg/mL for 24 h. After 48 h of incubation of A375 cells with free and liposomal CAPE, IC50 values were determined as 30.55 and 44.83 µg/mL, respectively. The flow cytometry analysis revealed that the apoptosis induced in A375 cancer cells was greater when treated with free CAPE than when treated with liposomal CAPE. The highest nanomechanical changes in the amount of cell adhesion forces, and elastic modulus value were seen in free CAPE. Subsequently, the greatest decrease in PI3K/AKT1 gene expression ratio occurred in free CAPE.