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1.
Anticancer Res ; 40(1): 221-227, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892570

RESUMO

BACKGROUND/AIM: Autophagy can be either tumor promotive or suppressive. We previously identified an autophagy-inducing activity in the 30-100 kDa fraction of areca-nut-extract (ANE 30-100K) and showed that several tumor cells subjected to chronic ANE 30-100K stimulation (CAS) exhibited higher resistance against stressed environments including serum-free (SF) conditions in vitro. Herein, we aimed to assess whether CAS can also provide growth advantages for tumor cells in vivo and the therapeutic effect of autophagy inhibition on CAS-treated tumors. MATERIALS AND METHODS: Esophageal CE81T/VGH cells and nude mice were used as experimental models. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ), as well as another anticancer drug cisplatin (DDP), were chosen to challenge CAS-treated CE81T/VGH cells in vitro and in vivo. RESULTS: CAS-treated CE81T/VGH cells expressed higher levels of microtubule-associated protein 1 light chain 3A/B-II (LC3-II) and beclin 1 proteins, and showed stronger resistance to SF and hypoxia conditions, that were mitigated by CQ or 3-MA in vitro. Furthermore, CAS-treated CE81T/VGH cells induced significantly larger tumors in mice, which were also attenuated by single 3-MA or CQ treatment. Finally, the combined treatment of 3-MA or CQ with DDP further up-regulated DDP-induced caspase-3 activity in vitro and exhibited synergistic anti-tumor effects on mice. CONCLUSION: CAS may up-regulate tumoral autophagy and provide growth advantage for tumors both in vitro and in vivo. Furthermore, autophagy inhibition alone or in combination with DDP may achieve positive therapy for tumors encountered with CAS.


Assuntos
Areca/química , Autofagia , Neoplasias/patologia , Nozes/química , Regulação para Cima , Animais , Autofagia/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus
2.
Nucleic Acids Res ; 47(19): 10212-10234, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31538203

RESUMO

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.


Assuntos
Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mapas de Interação de Proteínas/genética , Proteína Supressora de Tumor p53/genética , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Transdução de Sinais/genética
3.
Cell Physiol Biochem ; 53(3): 480-495, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486323

RESUMO

BACKGROUND/AIMS: Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. METHODS: HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. RESULTS: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. CONCLUSION: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.


Assuntos
Hipóxia Celular/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Western Blotting , Hipóxia Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células HCT116 , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células PC-3 , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
4.
Ann Clin Lab Sci ; 49(4): 457-467, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471334

RESUMO

This study aims to explore the regulatory mechanism of hypoxia-inducible factor HIF-1α on Kv3.4. Oral squamous cell carcinoma (OSCC) cell lines SCC3 and CAL27 were used in this study. Western blotting and qRT-PCR methods were used to detect Kv3.4 expression levels in OSCC and their adjacent tissues. The expression changes of Kv3.4 and HIF-1α in a hypoxic environment were detected in cell lines. The stable OSCC cell lines with knockouts of HIF-1α and Kv3.4 were constructed. Transwell and CCK-8 assays were used to detect changes in the invasion, migration and proliferation ability after transfection. Chromatin immunoprecipitation and luciferase reporter gene assays were used to determine the regulatory and binding sites of HIF-1α on Kv3.4. The expression level of Kv3.4 in oral cancer tissue was higher than normal oral epithelium's regular value. The expression level of HIF-1α and Kv3.4 increased under hypoxia. Knocking out HIF-1α and Kv3.4 could reduce the invasion, migration and proliferation of cells. A down regulation of HIF-1α will reduce the Kv3.4 expression level. Overexpressing Kv3.4 after knocking down HIF-1α partially restored the proliferation and invasion of cell lines. Therefore, HIF-1α regulates the invasion, migration and proliferation of oral cancer cells by regulating Kv3.4 expression.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Bucais/patologia , Canais de Potássio Shaw/metabolismo , Sequência de Bases , Hipóxia Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Neoplasias Bucais/genética , Invasividade Neoplásica , Regiões Promotoras Genéticas/genética , Ligação Proteica , Canais de Potássio Shaw/genética
5.
Anticancer Res ; 39(8): 4165-4170, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366501

RESUMO

AIM: To examine the influence of hypoxia on the in vitro growth of leukaemia cells and the activity of signalling proteins to better understand the pathophysiology of leukaemia cells in human bone marrow. MATERIALS AND METHODS: Six human leukaemia cell lines were cultured under normoxic or hypoxic conditions. Cell growth, recovery of clonogenic cells, and the expression and activation of various signalling proteins were examined. RESULTS: Hypoxia suppressed cell growth and the recovery of clonogenic cells. Moreover, hypoxia up-regulated hypoxia-inducible factor (HIF) 1α and HIF2α expression while suppressing the expression and activation of NOTCH1, mechanistic target of rapamycin kinase (mTOR) activation, and nuclear factor-kappa B (NF-κB) phosphorylation. CONCLUSION: We found that hypoxia up-regulated HIF expression while it suppressed the self-renewal capacity of leukaemia cells, NOTCH activity, and expression of its down-stream signalling molecules, which differs from previous reports mentioning that HIF activates NOTCH signalling. Our findings serve to further elucidate the in vivo pathophysiology of leukaemia cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia/genética , Receptor Notch1/genética , Ciclo Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia/patologia , NF-kappa B/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
6.
PLoS Genet ; 15(8): e1007980, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381576

RESUMO

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. Here, we report that, in the mutant for Trachealess (Trh), the Drosophila homolog for NPAS1 and NPAS3, smaller synaptic boutons form clusters named satellite boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the satellite bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. We further show that hypoxia-inducible factor (HIF)-1α/Similar (Sima) is critical in mediating hypoxia-induced satellite bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced satellite boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion.


Assuntos
Hipóxia Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Junção Neuromuscular/crescimento & desenvolvimento , Transmissão Sináptica/fisiologia , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Microscopia Intravital , Larva/fisiologia , Locomoção/genética , Microscopia Confocal , Microtúbulos/metabolismo , Modelos Animais , Neuroglia/citologia , Neuroglia/metabolismo , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Regulação para Cima , Proteína Wnt1/metabolismo
7.
Biomed Res Int ; 2019: 9174218, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31346526

RESUMO

Background: Intermittent hypoxia (IH) induced by obstructive sleep apnea (OSA) is a leading factor affecting cardiovascular fibrosis. Under IH condition, smooth muscle cells (SMAs) respond by dedifferentiation, which is associated with vascular remodelling. The expression of prolyl 4-hydroxylase domain protein 3 (PHD3) increases under hypoxia. However, the role of PHD3 in OSA-induced SMA dedifferentiation and cardiovascular fibrosis remains uncertain. Methods: We explored the mechanism of cardiovascular remodelling in C57BL/6 mice exposed to IH for 3 months and investigated the mechanism of PHD3 in improving the remodelling in vivo and vitro. Results: In vivo remodelling showed that IH induced cardiovascular fibrosis via SMC dedifferentiation and that fibrosis improved when PHD3 was overexpressed. In vitro remodelling showed that IH induced SMA dedifferentiation, which secretes much collagen I. PHD3 overexpression in cultured SMCs reversed the dedifferentiation by degrading and inactivating HIF-1α. Conclusion: OSA-induced cardiovascular fibrosis was associated with SMC dedifferentiation, and PHD3 overexpression may benefit its prevention by reversing the dedifferentiation. Therefore, PHD3 overexpression has therapeutic potential in disease treatment.


Assuntos
Cardiomiopatias/genética , Fibrose/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Apneia Obstrutiva do Sono/genética , Animais , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Desdiferenciação Celular/genética , Hipóxia Celular/genética , Modelos Animais de Doenças , Fibrose/etiologia , Fibrose/patologia , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Apneia Obstrutiva do Sono/complicações , Apneia Obstrutiva do Sono/patologia
8.
Artif Cells Nanomed Biotechnol ; 47(1): 3013-3020, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31321998

RESUMO

This research planned to dig the impacts and potential principles of long noncoding RNA RP4 onH9c2 cell injury induced by hypoxia. The H9c2 cardiac muscle cells were cultured under 3% O2 concentration to induce hypoxia injury, followed by detection of RP4 expression. RP4 was then overexpressed and silenced to investigate its effects on cell injury induced by hypoxia. The potential correlation between RP4 and miR-939, between miR-939 and Bnip3, and between RP4/miR-939/Bnip3 axis and Wnt/ß-catenin pathway activation were explored. Biological processes (suppressed cell viability, migration and invasion, but enhanced cell apoptosis) were changed by hypoxia. Upregulation of RP4 enhanced hypoxia-produced damage in H9c2 cells. Additionally, miR-939 expression was opposite regulated by RP4, and miR-939 mimic abrogated the influences of pc-RP4 on enhanced hypoxia damage in H9c2 cells. Moreover, Bnip3 was targeted by miR-939 and their correlation is negative. Furthermore, upregulation of RP4 exacerbated hypoxia-produced injury in H9c2 cells by sensitizing Wnt/ß-catenin signals in H9c2 cells, which was regulated by miR-939/Bnip3 axis. Our findings reveal that RP4 is highly expressed in the hypoxia-resulted H9c2 cells. Enhanced expression of RP4 may exacerbate hypoxia injury in cardiomyocytes through regulating miR-939/Bnip3 axis-mediated briskness of Wnt/ß-catenin signals. Our study will offer a fresh theoretical basis for the treatment of ischemic myocardial injury.


Assuntos
Proteínas de Membrana/metabolismo , MicroRNAs/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , Regulação para Cima , Via de Sinalização Wnt/genética , Animais , Hipóxia Celular/genética , Linhagem Celular , Ratos
9.
Artif Cells Nanomed Biotechnol ; 47(1): 2746-2753, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31282213

RESUMO

Puerarin has been reported to be useful in protection against hypoxia-induced injury. In our current study, we attempted to explore the protective effects of puerarin against hypoxia-caused damages in neural stem cells (NSCs). Additionally, the relative molecular underpinning studies preliminarily proceeded. NSCs were pre-incubated with puerarin before the hypoxic stimulus. MicroRNA-214 (miR-214) inhibitor was transfected into NSCs. Subsequently, the viability of NSCs was assessed by CCK-8 assay. Flow cytometry was employed to detect apoptotic cells after staining. qRT-PCR was performed to quantify miR-214. Western blot was applied for analyzing the expression of apoptosis-relative proteins and regulators. We found that puerarin alleviated hypoxia-induced apoptosis and maintained cell viability. Hypoxia-evoked up-regulation of miR-214 was further enhanced by puerarin. By contrast, miR-214-deficient NSCs showed the reduction in cell viability and the facilitation in apoptosis progress after pre-treatment with puerarin and stimulation in a hypoxia circumstance. Additionally, puerarin restored the phosphorylation of relative regulators, which was originally blunted by hypoxia. However, puerarin did not evidently restore the phosphorylation for response to hypoxia in miR-214-silenced NSCs. In conclusion, puerarin might be applied as a novel agent to ameliorate hypoxia-evoked damages in NSCs. Molecularly, miR-214 might be implicated in the protective roles of puerarin.


Assuntos
Isoflavonas/farmacologia , MicroRNAs/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citoproteção/efeitos dos fármacos , Citoproteção/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco Neurais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
10.
Artif Cells Nanomed Biotechnol ; 47(1): 2678-2687, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31257935

RESUMO

Emodin (EMO) possesses extensive pharmacological activities, which has been proven to exert the protective impact in diverse nervous system diseases. Nonetheless, whether EMO emerges a neuro-protective activity in hypoxic-evoked ischemic brain injury is still further probed. The intention of the research is to disclose whether EMO emerges neuro-protective activity in hypoxic-evoked ischemic brain injury. PC-12 received hypoxia administration, and then cell viability, apoptosis and autophagy were estimated. After EMO disposition, the above-involved cellular processes were evaluated again. MiR-25 functions in EMO-affected cells were also estimated. The interrelation between miR-25 and neurofilament light-chain polypeptide gene (NEFL) and the conceivable roles of NEFL in hypoxia-disposed cells were investigated. The latent mechanism was uncovered by mTOR and Notch pathways determination. Hypoxia triumphantly triggered apoptosis and autophagy, but EMO repressed these functions in PC-12 cells. Increased miR-25 was induced by EMO, and inhibited miR-25 abated the impacts of EMO on hypoxia-disposed PC-12 cells. NEFL as a neoteric target gene of miR-25 was predicated, and overexpressed NEFL annulled the functions of EMO in hypoxia-injured cells. EMO activated mTOR and Notch pathways through repressing NEFL. The investigations corroborated that EMO weakened hypoxia-triggered injury via elevating miR-25 by targeting NEFL in PC-12 cells.


Assuntos
Emodina/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Neurofilamentos/genética , Células PC12 , Ratos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
11.
Artif Cells Nanomed Biotechnol ; 47(1): 2221-2229, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31164009

RESUMO

Obstructive sleep apnoea-hypopnoea syndrome (OSAHS) is a condition causing apnea and hypopnea. LncRNA-ROR revealed properties in regulating hypoxia response. Our study explored the roles of ROR in CoCl2-induced hypoxia injury in HK-2 cells. HK-2 cells were treated with CoCl2 to induce hypoxia injury. Cell viability, cell apoptosis and apoptotic proteins were detected using CCK-8, flow cytometry and western blot, respectively. The alter expression of ROR and miR-145 was achieved through transfection. Moreover, the expressions of HIF-α and ERK and MAPK related factors were examined using western blot. We found that CoCl2 decreased cell viability and increased apoptosis as well as increased the expression of ROR. ROR overexpression increased cell viability, decreased cell apoptosis. ROR overexpression upregulated anti-apoptotic proteins Bcl-2 and decreased p53, Bax and cleaved-Caspase-3. ROR overexpression also increased the expression of HIF-α. On the opposite, ROR silence led to the opposite results as relative to ROR overexpression. ROR overexpression decreased expression of miR-145. Co-transfection with ROR overexpression and miR-145 impaired the promoting effects of ROR in CoCl2 treated cells. ROR increased phosphorylation of ERK while decreased phosphorylation of MAPK. In conclusion, lncRNA ROR alleviated CoCl2-induced hypoxia injury through regulation of miR-145 as well as modulating ERK and MAPK signalling. Highlights CoCl2 induces ROR upregulation; Overexpression of ROR reduces CoCl2-induced HK-2 cell injury; Silence of ROR promotes CoCl2-induced HK-2 cell injury; Overexpression of ROR decreases miR-145 expression; ROR overexpression modulates ERK and MAPK signalling pathways through regulation of miR-145.


Assuntos
Cobalto/farmacologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Inativação Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , RNA Longo não Codificante/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
12.
ACS Appl Mater Interfaces ; 11(29): 25740-25749, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31251022

RESUMO

The insufficient oxygen supply may cause hypoxia in a solid tumor, which can lead to drug resistance and unsatisfactory chemotherapy effect. To address this issue, a new nanodrug has been developed with azoreductase-responsive functional metal-organic frameworks (AMOFs), where chemotherapeutic drugs were encapsulated in the AMOFs and small interfering RNAs (siRNAs) were absorbed on the surface of AMOFs. The siRNA was designed to contain hypoxia-inducible factor (HIF)-1α against RX-0047, which can induce significant downregulation of HIF-1α protein. The azobenzene units within the frameworks of AMOFs could be reduced to amines by the highly expressed azoreductase under the oxygen-deficient environment, which results in azoreductase-responsive release of the encapsulated drugs and siRNAs under the hypoxic condition. Therefore, once the drug-loaded AMOF entered the hypoxic cancer cells, the azoreductase-responsive release of siRNA could decrease the efflux of chemotherapeutic drugs via inhibiting the expressions of HIF-1α, multidrug resistance gene 1, and P-glycoprotein. This nanodrug can thus efficiently break hypoxia-induced chemoresistance and result in high-efficient cancer therapy in hypoxic tumors. As far as we know, this is the first attempt to construct an AMOF-based nanodrug with hypoxic harvesting behaviors. This proof-of-concept research provides a simple strategy for the construction of hypoxic-responsive AMOFs and also offers a unique on-command drug delivery platform, which can effectively break hypoxia-induced chemoresistance.


Assuntos
Portadores de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , Nanoestruturas , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais , Oligonucleotídeos , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NADH NADPH Oxirredutases/genética , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Proteínas de Neoplasias/genética , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Int J Immunopathol Pharmacol ; 33: 2058738419857537, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31220954

RESUMO

Myocardial infarction (MI) is a serious heart disease in which cardiomyocytes are damaged, caused by hypoxia. This study explored the possible protective activity of Skullcapflavone I (SF I), a flavonoid isolated from the root of Scutellaria baicalensis Georgi, on hypoxia-stimulated cardiomyocytes cell injury in vitro. Viability and apoptosis of H9c2 cells and primary cardiomyocytes were tested using cell counting kit-8 (CCK-8) assay and Guava Nexin Reagent, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the long non-coding RNA regulator of reprogramming (lincRNA-ROR) expression. si-ROR was transfected to knockdown lincRNA-ROR. Western blotting was conducted to assess the protein levels of key molecules related to cell proliferation, apoptosis, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. We discovered that hypoxia stimulation obviously reduced H9c2 cell and primary cardiomyocytes' viability and proliferation, but promoted cell apoptosis. SF I treatment mitigated the cell viability and proliferation inhibition, as well as cell apoptosis caused by hypoxia. Moreover, SF I promoted the hypoxia-caused up-regulation of lincRNA-ROR in H9c2 cells and primary cardiomyocytes. Knockdown of lincRNA-ROR reversed the influence of SF I on hypoxia-stimulated H9c2 cells and primary cardiomyocytes. Besides, SF I activated MEK/ERK pathway in H9c2 cells and primary cardiomyocytes via up-regulating lincRNA-ROR. To sum up, our research verified the beneficial activity of SF I on hypoxia-caused cardiomyocytes injury. SF I protected cardiomyocytes from hypoxia-caused injury through up-regulation of lincRNA-ROR and activation of MEK/ERK pathway.


Assuntos
Cardiotônicos/farmacologia , Hipóxia Celular/genética , Flavonas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , RNA Longo não Codificante , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Ratos Wistar , Regulação para Cima/efeitos dos fármacos
14.
Eur J Pharmacol ; 857: 172449, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31207208

RESUMO

Endoplasmic reticulum stress (ERS)-induced cardiomyocyte apoptosis plays an important role in the pathological process following myocardial infarction (MI). Macrophages that express microRNA-155 (miR-155) mediate cardiac inflammation, fibrosis, and hypertrophy. Therefore, we investigated if miR-155 regulates ERS-induced cardiomyocyte apoptosis after MI using a mouse model, lipopolysaccharide (LPS)-induced rat bone marrow derived macrophages (BMDMs)and hypoxia-induced neonatal rat cardiomyocytes (NRCMs). In vivo, miR-155 levelswere significantly higher in the MI group compared to the sham group. MI increasedmacrophage infiltration, nuclear factor-κB (NF-κB) activation, ERS induced-apoptosis, and SOCS1 expression, all of which were attenuated by the miR-155 antagomir, with the exception of SOCS1 expression. Additionally, post-MI cardiac dysfunction was significantly improved by miR-155 inhibition. In vitro, LPS upregulated miR-155 expression in BMDMs, and the miR-155 antagomir decreased LPS-induced macrophage inflammation and NF-κB pathway activation, but increased expression of SOCS1. Hypoxia increased NF-κB pathway activation, ERS marker expression, and apoptosis in NRCMs. Interestingly, conditioned medium from LPS-induced macrophages in combination with the miR-155 antagomir decreased, while the miR-155 agomir increased, the hypoxia-induced effects in NRCM's. The miR-155 agomir effects were reversed by inhibiting the NF-κB pathway in cardiomyocytes. Moreover, SOCS1 knockdown in LPS-induced macrophages promoted NF-κB pathway activation and ERS-induced cardiomyocyte apoptosis in the hypoxia-induced NRCMs, but the SOCS1-siRNA-induced effects were markedly decreased by miR-155 antagomir treatment. These data suggest that miR-155 inhibition attenuates ERS-induced cardiomyocyte apoptosis after MI via reducing macrophage inflammation through the SOCS1/NF-κB pathway.


Assuntos
Apoptose/genética , Estresse do Retículo Endoplasmático/genética , Macrófagos/metabolismo , MicroRNAs/antagonistas & inibidores , Miócitos Cardíacos/patologia , Animais , Antagomirs/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Coração/fisiopatologia , Inflamação/genética , Inflamação/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , NF-kappa B/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína 1 Supressora da Sinalização de Citocina/genética
15.
Nat Med ; 25(5): 784-791, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31061540

RESUMO

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.


Assuntos
Lesões Encefálicas/etiologia , Hipóxia Encefálica/etiologia , Modelos Neurológicos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Hipóxia Encefálica/metabolismo , Hipóxia Encefálica/patologia , Lactente Extremamente Prematuro , Recém-Nascido , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/genética , Neurogênese/fisiologia , Organoides/metabolismo , Organoides/patologia , Proteínas com Domínio T/metabolismo , Resposta a Proteínas não Dobradas
16.
Cell Mol Biol Lett ; 24: 28, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061665

RESUMO

Background: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor with a pivotal role in physiological and pathological responses to hypoxia. While HIF-1α is known to be involved in hypoxia-induced upregulation of microRNA (miRNA) expression, HIF-1α is also targeted by miRNAs. In this study, miRNAs targeting HIF-1α were identified and their effects on its expression and downstream target genes under hypoxic conditions were investigated. Cell migration under the same conditions was also assessed. Methods: microRNAs that target HIF-1α were screened using 3'-untranslated region luciferase (3'-UTR-luciferase) reporter assays. The expression levels of HIF-1α and its downstream target genes after transfection with miRNA were assessed using quantitative RT-PCR and western blot analyses. The effect of the miRNAs on the transcriptional activity of HIF-1α was determined using hypoxia-responsive element luciferase (HRE-luciferase) assays. Cell migration under hypoxia was examined using the wound-healing assay. Results: Several of the 19 screened miRNAs considerably decreased the luciferase activity. Transfection with miR-200c had substantial impact on the expression level and transcription activity of HIF-1α. The mRNA level of HIF-1α downstream genes decreased in response to miR-200c overexpression. MiR-200c inhibited cell migration in normoxia and, to a greater extent, in hypoxia. These effects were partly reversed by HIF-1α expression under hypoxic conditions. Conclusion: miR-200c negatively affects hypoxia-induced responses by downregulating HIF-1α, a key regulator of hypoxia. Therefore, overexpression of miR-200c might have therapeutic potential as an anticancer agent that inhibits tumor hypoxia.


Assuntos
Movimento Celular/genética , Regulação para Baixo/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Hipóxia Celular/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Luciferases/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Regulação para Cima/genética , Cicatrização
17.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067669

RESUMO

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.


Assuntos
Mitocôndrias/metabolismo , Oócitos/metabolismo , Oogênese/genética , Transcriptoma , Animais , Hipóxia Celular/genética , Células Cultivadas , Feminino , Técnicas de Maturação in Vitro de Oócitos , Mitocôndrias/genética , Oócitos/citologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Suínos , Fator de Crescimento Transformador beta/metabolismo
18.
Mol Med Rep ; 20(1): 236-244, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115541

RESUMO

Pulmonary arterial hypertension (PAH) is a fatal and currently incurable cardiopulmonary disease. Numerous microRNAs (miRNAs) serve important roles in the development of PAH. While the expression of miR­30a­5p was downregulated in the lung tissue of rats in a pulmonary hypertension rat model, the expression pattern and function of miR­30a­5p in human PAH remain unclear. Reverse transcription quantitative polymerase chain reaction (RT­qPCR) was used to examine miR­30a­5p and chitinase­3­like protein 1 (YKL­40) mRNA expression levels. The expression levels of YKL­40 and apoptosis­associated proteins were measured by western blot analysis. Cell proliferation assays and flow cytometry analysis were performed to examine cell proliferation and apoptosis, respectively. The association between miR­30a­5p and YKL­40 was determined by a luciferase reporter assay, RT­qPCR and western blot analysis. The relative expression levels of miR­30a­5p in plasma were increased in patients with PAH [median=13.23 (25th percentile=6.388, 75th percentile=21.91)] compared with normal controls [median=2.25 (25th percentile=1.4, 75th percentile=3.7). The expression of miR­30a­5p was significantly downregulated while the protein expression of YKL­40 was significantly upregulated in hypoxia­induced human pulmonary artery endothelial cells (HPAECs) when compared with the hypoxia­induced group at 0 h. miR­30a­5p overexpression promoted HPAEC growth and inhibited apoptosis of HPAECs under hypoxia. A miR­30a­5p mimic decreased the luciferase activity of a luciferase reporter construct containing YKL­40 3'­untranslated region and also decreased YKL­40 protein expression. YKL­40 overexpression partly alleviated the effects of miR­30a­5p upregulation on proliferation and apoptosis of HPAECs under hypoxia. In conclusion, the data indicated that miR­30a­5p promoted cell growth and inhibited apoptosis of HPAECs under hypoxia by targeting YKL­40. Therefore, the miR­30a­5p/YKL­40 axis may provide a potential target for the development of novel PAH therapies.


Assuntos
Proteína 1 Semelhante à Quitinase-3/genética , Hipertensão Pulmonar/genética , MicroRNAs/genética , Animais , Apoptose/genética , Hipóxia Celular/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Hipertensão Pulmonar/patologia , Pulmão/metabolismo , Pulmão/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos
19.
Mol Med Rep ; 20(1): 350-358, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115545

RESUMO

Endoplasmic reticulum (ER) stress and autophagy are involved in myocardial ischemia­reperfusion (I/R) injury; however, their roles in this type of injury remain unclear. The present study investigated the roles of ER stress and autophagy, and their underlying mechanisms, in H9c2 cells during hypoxia/reoxygenation (H/R) injury. Cell viability was detected by CCK­8 assay. The autophagy flux was monitored with mCherry­GFP­LC3­adenovirus transfection. The expression levels of autophagy­related proteins and ER stress­related proteins were measured by western blotting. Apoptosis was detected by flow cytometry and western blotting. The results indicated that autophagy was induced, ER stress was activated and apoptosis was promoted in H9c2 cells during H/R injury. The inhibition of ER stress by 4­phenylbutyrate or C/EBP homologous protein (CHOP)­targeting small interfering RNA (siRNA) decreased autophagy and ameliorated cell apoptosis during H/R injury. Activation of autophagy by rapamycin attenuated ER stress and ameliorated cell apoptosis. Inhibition of autophagy by 3­methyladenine or Beclin1­targeting siRNA aggravated ER stress and exacerbated cell apoptosis, and activation of ER stress by thapsigargin decreased autophagy and induced cell apoptosis. Collectively, the findings of the present study demonstrated that H/R induced apoptosis and autophagy via ER stress in H9c2 cells, and that CHOP may serve an important role in ER stress­induced autophagy and apoptosis. Autophagy, as an adaptive response, was activated by ER stress and alleviated ER stress­induced cell apoptosis during H/R injury.


Assuntos
Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteína Beclina-1/genética , Hipóxia Celular/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenilbutiratos/farmacologia , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/genética
20.
Biomed Pharmacother ; 116: 108836, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004838

RESUMO

BACKGROUND: An impaired trophoblast invasion ability contributes to the development of pre-eclampsia (PE), and can be induced by the altered expression of various microRNAs (miRs). MiR-141 and CXCL12ß (C-X-C motif chemokine ligand 12) signaling regulate trophoblast invasion and vascularization capabilities during PE pathogenesis; however, their interactions and underlying mechanisms of action remain unclear. We investigated how miR-141 modulates trophoblast invasion, with a focus on its interaction with CXCL12ß signaling. METHODS: A PE model was established by using HTR-8/SVneo cells, which were first cultured with 2% O2 for 48 h, and then with 5% O2. The expression of miR-141 in human villous trophoblast HTR-8/SVneo cells was modulated with mimics or an inhibitor, and analyzed by quantitative RT-PCR. CXCL12ß levels were determined by ELISA. Cell apoptosis was determined by flow cytometry, and the invasion and vascularization capabilities of trophoblasts were evaluated by Transwell and tube formation assays, respectively. Binding of miR-141 with CXCL12ß mRNA was verified by the dual luciferase assay. Protein levels were estimated by western blotting. RESULTS: MiR-141 expression was significantly induced by hypoxia in HTR-8/SVneo cells. MiR-141 was found to promote apoptosis and inhibit the invasion and vascularization abilities of HTR-8/SVneo cells under conditions of hypoxia. MiR-141 could directly bind with the 3'UTR region of CXCL12ß mRNA and inhibit its translation. In addition, we proved that miR-141 could inhibit the invasion and vascularization abilities, and promote the apoptosis of HTR-8/SVneo cells by targeting CXCL12ß under hypoxic conditions. Furthermore, we demonstrated that arachidonic acid could reverse the invasion and apoptosis abilities of HTR-8/SVneo cells mediated by CXCL12ß during hypoxia. In terms of mechanism, MiR-141 could downregulate MMP2, p62, and LC3B expression, and upregulate ROCK1 and RhoA expression in HTR-8/SVneo cells by targeting the CXCL12ß gene during hypoxia. The effects of CXCL12ßon HTR-8/SVneo cells could be reversed by arachidonic acid (ARA). CONCLUSION: Induction of miR-141 by hypoxia promotes apoptosis, and inhibits the invasion and vascularization capabilities of HTR-8/SVneo cells by suppressing the CXCL12ß and CXCR2/4 signaling pathways.


Assuntos
Apoptose , Quimiocina CXCL12/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Trofoblastos/patologia , Apoptose/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Sequência de Bases , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , MicroRNAs/genética , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo
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